Allosteric receptor modulation in drug targeting

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© 2006 by Taylor & Francis Group, LLC

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edited by

Norman G. Bowery University of Birmingham Birmingham, U.K. GlaxoSmithKline Verona, Italy

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Published in 2006 by Taylor & Francis Group 270 Madison Avenue New York, NY 10016 © 2006 by Taylor & Francis Group, LLC No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-2791-6 (Hardcover) International Standard Book Number-13: 978-0-8247-2791-8 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe.

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Preface Preface

Allosteric modulation of receptors for endogenous ligands has, in recent years, attracted considerable interest from the drug research community. There may be a number of reasons for this but not least of these must surely be the success of the benzodiazepine class of anxiolytic/sedative drugs. These positive receptor modulators were ﬁrst introduced at the end of the 1960s in response to the need for safer sedatives that would not produce the respiratory depression associated with the action of the existing barbiturate class of drugs. The benzodiazepines allosterically modulate the ionotropic GABAA receptor to enhance the action of the inhibitory transmitter GABA. In the absence of GABA the benzodiazepines are inert, which is a feature of allosteric drug action. The allosteric agent requires the presence of the receptor agonist in order to exert its effect. This characteristic can be of considerable beneﬁt over directly acting receptor agonists that act at orthosteric sites. Excessive activation of the receptor system cannot occur because the maximal stimulation that can arise is dependent upon the concentration of the natural receptor ligand. This, in turn, is less likely to produce receptor desensitisation or tolerance as can occur with full receptor agonists. So what does the term ‘‘allosteric modulator’’ mean? Neubig and colleagues (2003, Pharmacol Rev, 55, 597–606) have deﬁned this term, under the auspices of the International Union of Pharmacology, as ‘‘a ligand that increases or decreases the action of an agonist or antagonist by combining with a distinct site on the receptor macromolecule.’’ Thus, in the example of the benzodiazepines there exist distinct receptors on the GABAA receptor macromolecule that on activation increase the maximal response to the agonist, GABA, as well as increasing the apparent sensitivity to it. The concept of allosterism in drug action was introduced more than 40 years ago (Monod et al. 1963;1965, J Mol Biol 6, 306–329, 12, 88–118 ) at a time when the structures of receptors were beginning to emerge. The original iii

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focus of interest in allosterism was associated with ionotropic cholinoceptors. This association then expanded to encompass metabotropic as well as other ionotropic receptors, such that there are now many examples to provide the potential for application to therapeutics. The obvious application is in agonist enhancement but negative modulation is also a possibility though examples of this are relatively uncommon. This volume provides some examples of the receptors where allosteric activity has been deﬁned including ionotropic GABAA, 5-HT3, glutamatergic and nicotinic receptors, as well as metabotropic G-protein–coupled receptors such as mGluR, muscarinic, GABAB, and a-adrenoceptors. However, before considering these examples, it is important that the underlying features, principles, and characteristics of allosteric mechanisms are described. As a consequence, the ﬁrst section comprises three contributions that provide these details as well as modelling allosteric action. Stephen Daniels, Terry Kenakin, and David Hall, all of whom are recognized experts in the principles of drug action, have prepared these chapters. Among the many examples of allosteric modulation included in this volume are the benzodiazepines and the coverage given by Hanns Mo¨hler in section 2 provides up-to-date information on their characteristics. Subsequent chapters in this section are focused on the other ionotropic receptors within the same structural superfamily. Section 3 is devoted to G-protein coupled receptors (GPCRs) and commences with an introductory chapter by Ad Ijzerman and colleagues on the general concept of allosterism at GPCRs. This is followed by comparative information on receptor examples. While the information is not exhaustive it is hoped that sufﬁcient is provided to enable the reader to gain a clear understanding of their comparative features. There is no doubt that receptor allosterism is and will continue to become more important in the quest to ﬁnd drug targets for a variety of diseases. Perhaps the material included in this volume will provide the background data required to facilitate this process. Norman G. Bowery

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Contents

Preface . . . . iii Contributors . . . . xi SECTION I:

BASIC PRINCIPLES

1. Allosteric Modulation of Receptor Function . . . . . . . . . . . . 1 Stephen Daniels The Advantages of Allosteric Compared to Orthosteric Modulation . . . . 2 The Detection of Allosteric Function . . . . 2 Allosteric Modulation of Ionotropic Receptors . . . . 3 Allosteric Modulation of Metabotropic Receptors . . . . 9 Conclusions . . . . 13 References . . . . 14 2. Characteristics of Allosterism in Drug Action . . . . . . . . . . 19 Terry P. Kenakin Global Protein Perturbation . . . . 19 Practical Aspects of Allosteric Probe Dependence . . . . 22 Probe-Dependent Antagonism . . . . 24 Unique Properties of Allosteric Ligands . . . . 25 The Detection and Quantiﬁcation of Allosteric Effect . . . . 28 The Future of Allosteric Ligands as Drugs . . . . 33 References . . . . 33

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3. Predicting Dose–Response Curve Behavior . . . . . . . . . . . . David A. Hall Introduction . . . . 39 Modeling Allosteric Effects on Ligand Binding . . . . 43 Modeling the Functional Effects of Allosteric Ligands . . . . 53 Summary . . . . 67 Appendix . . . . 70 References . . . . 75 SECTION II:

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IONOTROPIC RECEPTORS

4. Allosteric Modulation of GABAA Receptors . . . . . . . . . . . 79 Hanns Mo¨hler Introduction . . . . 79 GABAA Receptors as Allosteric Proteins: Bidirectional Modulation . . . . 80 Synaptic Mechanism of Allosteric Action at GABAA Receptors . . . . 81 Partial Bidirectional Modulators of GABAA Receptors . . . . 83 Antagonist of Allosteric Modulation . . . . 83 GABAA Receptor Subtypes: A New Allosteric Pharmacology . . . . 84 Allosteric Modulation of Sleep . . . . 86 Allosteric Anxiolysis . . . . 86 Allosteric Enhancement of Learning and Memory . . . . 88 Allosteric Modulation of Consciousness . . . . 88 Conclusions . . . . 89 References . . . . 89 5. Allosteric Interactions at the NMDA Receptor Channel Complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Manolo Mugnaini Introduction . . . . 93 Allosteric Sites of NMDA Receptors . . . . 100 Other Substances Modulating NMDA Receptor Function . . . . 115 Therapeutic Potential of Allosteric Modulators of NMDA Receptors . . . . 116

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Concluding Remarks . . . . 118 References . . . . 119 6. 5-HT3 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Li Zhang and Sarah C. R. Lummis Introduction . . . . 135 5-HT3 Receptor Pharmacology . . . . 136 Receptor Structure . . . . 137 Receptor Subtypes . . . . 140 Distribution . . . . 140 Posttranslational Modiﬁcations . . . . 141 Allosteric Modulators . . . . 142 Therapeutic Potential . . . . 145 Conclusion . . . . 146 References . . . . 147

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7. Nicotinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 155 R. C. Hogg and D. Bertrand The nAChR as a Prototype of an Allosteric Protein . . . . 155 Receptor Modulation by Allosteric Ligands . . . . 159 The a7 Model . . . . 160 Inﬂuence of Receptor Subunit Composition on Receptor Properties . . . . 161 Allosteric Modulators . . . . 162 Positive Allosteric Effectors . . . . 164 Negative Allosteric Modulation of the nAChR . . . . 169 Conclusions . . . . 172 References . . . . 172 SECTION III:

G-PROTEIN–COUPLED RECEPTORS

8. Allosteric Modulation of G-Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Willem Soudijn, Ineke van Wijngaarden, and Ad P. Ijzerman Introduction . . . . 179 Speciﬁc Examples of Allosteric Modulators . . . . 181 Clinical Studies . . . . 199 Concluding Remarks . . . . 199 References . . . . 202

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9. Allosteric Modulators of Group III Metabotropic Glutamate Receptors as Novel Therapeutics . . . . . . . . . . . . . . . . . . 207 Jesper Mosolff Mathiesen and M. Teresa Ramirez Metabotropic Glutamate Receptors in Glutamatergic Neurotransmission . . . . 207 Role of Group III mGluRs in CNS Disorders . . . . 212 Models of mGluR Allosteric Modulation . . . . 214 Groups I and II Allosteric Modulators . . . . 216 Group III mGluR Allosteric Modulators . . . . 218 Potential Mechanistic Effects of a Group III Positive Allosteric Modulator . . . . 227 Perspective . . . . 229 References . . . . 230

10. Allosteric Modulation of GABAB Receptors . . . . . . . . . . Stephan Urwyler Introduction: Structure and Function of the GABAB Receptor . . . . 235 The Discovery of Allosteric GABAB Receptor Modulators . . . . 239 Effects of Allosteric Modulators at Native GABAB Receptors . . . . 241 Molecular Mechanisms and Site of Action of Allosteric GABAB Receptor Modulation by CGP7930 and GS39783 . . . . 241 Theoretical Aspects of Allosteric Modulation; Effects of Modulators on Orthosteric Ligands with Distinct Intrinsic Efﬁcacies . . . . 244 GABAB Receptor Modulation in Cellular and Physiological Assay Systems . . . . 244 Enhancement of GABAB Receptor Function by Other Mechanisms and Other Agents . . . . 246 Effects of Allosteric GABAB Receptor Modulators In Vivo . . . . 248 Outlook: Possible Therapeutic Applications of Positive GABAB Receptor Modulators and Future Prospects . . . . 250 References . . . . 251

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11. Allosteric Interactions at GABAB and Related G-Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . 259 David I. B. Kerr and Jennifer Ong Introduction . . . . 259 Origin of Family 3 GPCRs . . . . 260 Allosteric Modulators for Family 3 GPCRs . . . . 261 Modulators at mGluRs . . . . 262 Calcium-Sensing Receptors . . . . 264 Allosteric Modulation at GABAB Receptors . . . . 266 Calcium Positively Modulates GABAB and mGlu Receptors . . . . 267 CGP 7930 and GS 39783 Are Allosteric Modulators at GABAB Receptors . . . . 267 Proposed Site of Action of Arylalkylamines at GABAB Receptors . . . . 269 L-Amino Acids Potentiate Baclofen Responses in Rat Neocortical Slices . . . . 270 Positive Allosteric Actions of Amino Acids at Recombinant GABAB Receptors . . . . 271 L-Gln, L-Asn and L-Orn Are Also Potent Positive Modulators of GABAB Receptors . . . . 271 Hyperpolarizing Effects of Amino Acids in Rat Neocortical Slices . . . . 273 Interactions Between Amino Acids and Sch 50911 . . . . 274 Allosteric Interactions at Family 3 GPCRs . . . . 275 Allosteric Interactions at GABAB Receptors . . . . 276 Summary and Conclusions . . . . 277 References . . . . 278 12. Muscarinic Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Christian Tra¨nkle Introduction . . . . 287 Principles of Muscarinic Allosteric Action . . . . 288 Muscarinic Receptor Speciﬁcity of Allosteric Actions . . . . 292 Search for a Potential Allosteric Radioligand of M2 Receptors . . . . 293 Binding Topology of Allosteric Modulators in Muscarinic M2 Receptors . . . . 296

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Muscarinic Subtype Selectivity of Dimethyl-W84 . . . . 297 Functional Effects of Dimethyl-W84 in M2 Receptors . . . . 300 [3H]Dimethyl-W84 as a Radioligand for the Common Allosteric Binding Site of Muscarinic M2 Receptors . . . . 301 Use of the Radioalloster [3H]Dimethyl-W84 to Test Predictions of the Cooperativity Model for the Binding of Allosteric Modulators at the Common Allosteric Binding Site of M2 Receptors . . . . 304 Interactions of Allosteric and Orthosteric Ligands with [3H]Dimethyl-W84 at the Common Allosteric Site of Muscarinic M2 Receptors . . . . 306 Common Site Receptor Epitopes Identiﬁed by Site-Directed Mutagenesis as a Basis for a M2 Receptor Pharmacophore Model Comprising Dimethyl-W84 . . . . 313 Concluding Remarks . . . . 316 References . . . . 317 13. a2-Adrenoceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Emma S. J. Robinson and Alan L. Hudson Introduction . . . . 327 Subtypes of a2-Adrenoceptors . . . . 329 a2-Adrenoceptor Localization and Subtype Distribution in the CNS . . . . 329 a2-Adrenoceptor-Mediated Functions in the CNS . . . . 331 a2-Adrenoceptors in Neurological and Psychiatric Disorders . . . . 335 a2-Adrenoceptors and Allosteric Interactions . . . . 338 Ionic Modulation and Effects of Amiloride . . . . 343 Future Prospects of Allosteric Modulation of a2-Adrenoceptors . . . . 344 References . . . . 344

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Contributors

D. Bertrand Department of Neuroscience, Centre Me´dical Universitaire, Geneva, Switzerland Stephen Daniels Cardiff, U.K.

Welsh School of Pharmacy, Cardiff University,

David A. Hall Respiratory Pharmacology, Respiratory and Inﬂammation Center of Excellence for Drug Discovery, GlaxoSmithKline, Stevenage, Herts, U.K. R. C. Hogg Department of Neuroscience, Centre Me´dical Universitaire, Geneva, Switzerland Alan L. Hudson Psychopharmacology Unit, University of Bristol, Bristol, U.K. and Department of Pharmacology, Medical Sciences Building, University of Alberta, Edmonton, Canada Ad P. Ijzerman Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands Terry P. Kenakin Assay Development, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina, U.S.A. David I. B. Kerr Department of Anaesthesia and Intensive Care, The University of Adelaide, Adelaide, South Australia, Australia

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Sarah C. R. Lummis Department of Biochemistry, University of Cambridge, Cambridge, U.K. Jesper Mosolff Mathiesen Department of Molecular Pharmacology, H. Lundbeck A/S, Valby and Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark Hanns Mo¨hler Department of Chemistry and Applied Biosciences, Institute of Pharmacology, University of Zurich, Federal Institute of Technology (ETH) and Collegium Helveticum, Zurich, Switzerland Manolo Mugnaini Biology Department, Psychiatry Center of Excellence for Drug Discovery, GlaxoSmithKline Medicines Research Center, Verona, Italy Jennifer Ong Department of Anaesthesia and Intensive Care, The University of Adelaide, Adelaide, South Australia, Australia M. Teresa Ramirez Department of Molecular Pharmacology, Zealand Pharma A/S, Glostrup, Denmark Emma S. J. Robinson Department of Pharmacology, School of Medical Sciences, University Walk, University of Bristol, Bristol, U.K. Willem Soudijn Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands Christian Tra¨nkle Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany Stephan Urwyler Department of Neuroscience, Novartis Institutes for BioMedical Research, Basel, Switzerland Ineke van Wijngaarden Division of Medicinal Chemistry, Leiden/ Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands Li Zhang Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, U.S.A.

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SECTION I: BASIC PRINCIPLES

1 Allosteric Modulation of Receptor Function Stephen Daniels Welsh School of Pharmacy, Cardiff University, Cardiff, U.K.

Cell surface receptors have long been a target for drug development with the intent to modulate receptor-mediated signalling to correct a pathophysiological state or provide symptomatic relief. However, traditional agonist, antagonist, or channel-blocking drugs are frequently associated with a high incidence of side effects and the development of tolerance and dependence. The binding site for the endogenous agonist is likely to be a highly conserved structural region, for a given class of receptor [e.g., the c-aminobutyric acid type A receptors (GABAA-Rs)] and therefore unlikely to allow great selectivity, in agonist or antagonist activity, between different subtypes of a receptor class. This is probably one of the reasons for the difﬁculty in establishing a clinical role for glutamate receptor antagonists (e.g., CPPene) for the treatment of various neurological disorders (e.g., epilepsy, stroke), where despite pharmacological efﬁcacy, the side effect proﬁle precludes their use (1). Similarly, the ion channel associated with the ionotropic receptors is also likely to be highly conserved between different subtypes of a particular receptor class. This again offers little opportunity for selectivity and the likelihood of an unacceptable side effect proﬁle.

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THE ADVANTAGES OF ALLOSTERIC COMPARED TO ORTHOSTERIC MODULATION As an alternative, drugs that act at a site spatially distinct from that at which the endogenous agonist acts, an allosteric as opposed to an orthosteric site, may overcome these problems. The benzodiazepines, which enhance the activity of GABAA receptors, are a classic example of such allosteric modulators (2). Allosteric modulators offer a number of advantages over conventional agonist or antagonist drugs. First, they frequently exhibit little or no intrinsic activity since their mode of action is to enhance or inhibit the action of the endogenous agonist. In this respect, they only elicit an effect in the tissue(s) stimulated by the endogenous agonist; an effect which is also in synchrony with the frequency of the physiological stimulation. In principle, this should reduce the likelihood of the target receptor desensitizing, even in the continued presence of the allosteric modulator, thus removing one of the mechanisms for acquired tolerance. Second, the action of an allosteric modulator is saturable; i.e., once the allosteric sites are fully occupied, no further allosteric effect is observed (3). Allosteric drugs should, therefore, be safer under conditions of overdose than conventional orthosteric drugs. The third signiﬁcant attraction of allosteric drugs is the possibility that an effective allosteric site could be found which is speciﬁc to one subtype of a receptor class because they can be targeted at nonconserved sites. Thus, benzodiazepines act as positive (diazepam, ﬂunitrazepam) or negative (ﬂumazenil) allosteric modulators at the a1,2,3,5bnc2 but are without effect at a4,6bnc subtypes of the GABAA receptor (2). In addition to allosteric site deﬁnition, it is possible that drugs may bind to an allosteric site but fail to express any cooperative effect (either positive or negative). This is exempliﬁed by the action of N-chloromethyl brucine at muscarinic acetylcholine receptors (mACh-Rs); it is a positive allosteric modulator at M3, a negative allosteric modulator at M2, and is without effect at M1 or M4 subtypes of the mACh receptor, despite demonstrating equivalent binding afﬁnity (4). N-chloromethyl brucine has been termed a ‘‘neutral’’ allosteric modulator at the M1,4 subtypes of the mACh receptor. Finally, allosteric modulators may be effective at various peptide and hormone G-protein coupled receptors in which the topographical arrangement of the orthosteric site makes it difﬁcult for a small molecule to mimic the endogenous ligand (5).

THE DETECTION OF ALLOSTERIC FUNCTION Until recently the recognition of allosteric modulators in drug discovery programs has been hampered by the almost universal use of equilibrium

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radioligand binding assays in high-throughput screening systems. Although an allosteric modulator may alter binding at the orthosteric site (6,7), the use of an inappropriately high concentration of the orthosteric ligand may mask the allosteric effect or, in the case of negative allosteric modulators, may cause the allosteric interaction to resemble antagonism (3). As an alternative to equilibrium binding assays it is possible to use radiolabeled techniques to measure the rate of association/dissociation of the orthosteric ligand. Such measurements are frequently more successful at detecting allosteric interactions than equilibrium binding methods (8). However, functional assays are far more likely to reliably detect allosteric interactions and, with the advent of a variety of different techniques (reporter systems, yeast and melanophore systems, ﬂuorescence-based intracellular calcium measurements), are becoming adapted for high-throughput screening. Such methods are clearly capable of demonstrating allosteric interactions when nonequilibrium radiolabeled binding methods fail (9). Nevertheless, there can be difﬁculties, even with functional assays, in deﬁning the receptor selectivity of an allosteric effect. The endogenous fatty acid, oleamide, can activate 5-hydroxytryptamine receptors type 7 (5-HT7), expressed in HeLa cells, in the absence of the orthosteric agonist 5-HT via an allosteric mechanism (10,11). However, the 5-HT7 selective antagonist, clozapine, failed to inhibit the oleamide effect, although the oleamideinduced signalling was not seen in cells not transfected with 5-HT7 receptors. It would seem therefore that to maximize the likelihood of detecting allosteric effects, including inverse agonism, a functional assay is necessary. There are disadvantages, including difﬁculties in ascribing effects to a speciﬁc receptor or the activation of nonreceptor-mediated signalling, but these may be offset by the judicious use of radiolabeled binding methods as secondary screens (3).

ALLOSTERIC MODULATION OF IONOTROPIC RECEPTORS Anxiolytics The GABAA receptor has binding sites for many neuroactive substances including barbiturates, benzodiazepines, convulsants, general anesthetics, and neurosteroids. Of these, the benzodiazepines have been recognized as classic allosteric modulators and they have been in widespread clinical use since the early 1960s as anxiolytics and to treat insomnia. There is a wide spectrum of benzodiazepines including full agonists with varying pharmacodynamic and pharmacokinetic properties (e.g., diazepam, ﬂunitrazepam, lorazepam), partial agonists (e.g., bretazenil, imidazenil), inverse agonists (e.g., methyl-6,7-dimethoxy-4-ethyl-b-carboline-3-carboxylate) and partial inverse agonists (e.g., N-methyl-b-carboline-3-carboxamide) that are anxiogenic and either convulsant or proconvulsant and full antagonists

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(e.g., ﬂumazenil) that are without effect (12–14). It is now known that the benzodiazepines bind to the GABAA receptor and increase the number of channel openings when GABA is bound (full/partial agonists), which causes an increased chloride ﬂux into the cell that results in a hyperpolarization of the resting membrane potential and hence a reduced likelihood of triggering an action potential (15,16). Conversely, the inverse agonists decrease the number of channel openings when GABA is bound, depolarizing the cell and increasing excitability. The antagonists have no effect on channel opening and thus do not affect the resting membrane potential. Despite the widespread clinical use of the benzodiazepines it became clear that they suffer a number of drawbacks. Acutely, they induce sedation and cognitive dysfunction and chronically they produce tolerance and both physical and psychological dependence with patients suffering severe withdrawal effects (14). In consequence their use has declined in recent years and there is a general guidance that their use should be restricted to the short-term (less than 4 weeks). It was hoped that the partial agonists would provide the anxiolytic effect without the sedation and dependence associated with chronic use of the full agonists. This, however, is not the case. There has therefore been much recent interest in establishing exactly where the different benzodiazepines bind and whether it would be possible to develop subtype selective agents that would have anxiolytic properties but without the sedation and dependence. Recent experiments using molecular genetic techniques have begun to establish the benzodiazepine binding sites and to dissect the GABAA receptor subunits involved in sedation, anxiety, amnesia, and convulsive activity (2,13,17). The classic benzodiazepines (diazepam, ﬂunitrazepam) bind to GABAA receptors that comprise a1, a2, a3, or a5 subunits in combination with any b and c2 subunits. This receptor population accounts for approximately three-quarters of the total GABAA receptor population (13). GABAA receptors containing a4 or a6 subunits are insensitive to benzodiazepines. Mice having GABAA receptors containing a1 subunits that have been rendered insensitive to diazepam (by site-directed mutagenesis of a histidine residue for an argentine in the a subunit) show little sedation, and this was shown to be speciﬁc to ligands binding at the benzodiazepine site because barbiturates and neurosteroids were still as effective as in wild type mice (2). In similar experiments, the a1 subunit was shown to be associated with anterograde amnesia, a signiﬁcant side effect of benzodiazepines. The anticonvulsant activity of diazepam was tested against pentylenetetrazoleinduced seizures in mice expressing benzodiazepine insensitive a1 subunits, and it was shown that the anticonvulsant properties of diazepam are only partially expressed through the a1 containing GABAA receptors. In contrast, the anticonvulsant properties of the a1-selective imidazopyridine, zolpidem, are wholly mediated through its a1/c2 binding (2). The a5 subunit appears to regulate cognitive processes, (13). There are data that suggest that the a2 subunit regulates anxiety (2); however, more

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recently, Atack et al. (2) have provided evidence that the a3 subunit also mediates anxiety. At present there are no satisfactory a2 and a3 selective ligands which will restrict further progress towards achieving the target of a benzodiazepine which does not cause sedation, impair cognitive processes, and induce dependence. It should be noted that approximately 25% of GABAA receptors contain mixed a subunit types (e.g., a1a6bc2, a1a3bc2) and these receptors will display pharmacology which reﬂects the binding characteristics of each a subunit. This may make the search for the ideal benzodiazepine ligand more difﬁcult. General Anesthetics The search for a molecular mechanism that would explain how general anesthetics induce a reversible loss of consciousness has lasted over 100 years. Despite considerable progress in recent years, in line with the general increase in understanding the regulation of cellular signalling through the combined application of functional studies with molecular biological and genetic techniques, we are still far from being able to deﬁne the essential pharmacology required to induce anesthesia. Thus, even the ‘‘best’’ modern anesthetics in clinical use (sevoﬂurane, desﬂurane, propofol) coupled with ‘‘best practice’’ in modern anesthesia still have the capacity to induce a fatal overdose with doses only three to four times greater than that necessary for anesthesia. General anesthetics are capable of acting as allosteric modulators of all members of the cys-loop group of ionotropic receptors nicotinic acetylcholine (nACh), 5-HT3, GABAA, glycine). However, their actions are not always consistent nor necessarily linked to their potency as general anesthetics. Thus, neuronal a7 and muscle abcd nACh receptors, which are inhibited by thiopentone, do not differentiate in potency between the optical isomers of thiopentone, and the neuronal a4b2 receptor shows the opposite potency ratio between the optical isomers compared to that observed for anesthetic potency (18). Volatile anesthetics potentiate heteromeric neuronal nACh receptors (e.g., a3b4) at concentrations much less than those which produce general anesthesia (18,19), while neuromuscular junction (abcd) and neuronal a7 receptors are relatively unaffected by volatile anesthetics. Although the exact mechanism by which volatile anesthetics modulate heteromeric neuronal nicotinic receptors, the extracellular loop linking M2 and M3 transmembrane segments appears vital (20). Heteromeric neuronal nACh receptors may, therefore, play a part in regulating via an allosteric mechanism the action of volatile, but not intravenous, anesthetics. Volatile anesthetics potentiate currents recorded from recombinant 3-HT3 receptors whereas many intravenous anesthetics inhibit 5-HTstimulated [C14]guanidinium inﬂux into NIE/115 neuroblastoma cells (21). This latter effect may arise from a noncompetitive antagonism rather than via an allosteric inhibition.

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Perhaps of more relevance to general anesthesia are the actions of general anesthetics at GABAA and glycine receptors. The great majority of the GABAA receptor subtypes are potentiated by volatile anesthetics, and this activity correlates well with their anesthetic potency (22). The intravenous anesthetics (propofol, barbiturates, etomidate, and the anesthetic steroid 5a-pregnan-3a-ol-20-one) all potentiate GABAA receptors (23). Glycine receptors are also potentiated by barbiturates and propofol but not etomidate or 5a-pregnan-3a-ol-20-one (23). Glycine receptors are also potentiated by volatile anesthetics (24,25). The gaseous anesthetics xenon and nitrous oxide have no appreciable effect on GABAA receptors, but do potentiate glycine receptors (25–27). The identiﬁcation of a subset of GABAA receptors as being implicated in a mechanism linked to sedation led to an elegant set of experiments in which the action of etomidate, which is selective for receptors containing b2 and b3 subunits, was tested in genetically modiﬁed mice having etomidate-insensitive b2 subunits (28). It was concluded that while the b3 subunit was associated with etomidate’s anesthetic potency, the hypnotic and sedative properties were mediated via different mechanisms. The authors proposed that the ‘‘hypothesis of anesthesia producing a general global depression should be revised.’’ This certainly appears to be the case, and while the cys-loop ionotropic receptors are important targets for general anesthetics, allosteric interactions at these receptors are far from the complete story. The ionotropic glutamate receptors N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA), named after their selective agonists, are in general insensitive to general anesthetics. Those that do have effect, such as ketamine and xenon, appear to do so through noncompetitive mechanisms rather than allosteric mechanisms (29,30). It would seem that: simple gaseous anesthetics (xenon, nitrous oxide) act by allosteric potentiation of glycine receptors, and as noncompetitive antagonists of glutamate receptors; volatile anesthetics act as positive allosteric modulators of many subtypes of GABAA receptors, glycine receptors, 5-HT3 receptors, and heteromeric neuronal nACh receptors; and intravenous anesthetics (propofol, barbiturates, steroids) act primarily as positive allosteric modulators at GABAA receptors. It is probable that these actions do not wholly deﬁne the anesthetic properties of these substances and that a more complex pattern of neuronal modiﬁcation is involved. This awaits further experimental dissection but the necessary tools now appear to be available. Neurosteroids Neuroactive steroids are synthesized in the brain and act to regulate neuronal excitability via modulation of cell-surface receptors (31), in contrast to the classical genomic activity of steroids to regulate gene expression. The

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3a-reduced metabolites of progesterone and deoxycorticosterone (e.g., 3a, 5a-tetrahydroprogesterone; 3a,5a-tetrahydrodeoxycorticosterone), pregnenolone sulfate, and dehydroepiandrosterone sulfate are positive allosteric modulators of speciﬁc neurotransmitter receptors, especially the GABAA receptor. More recently, evidence has accumulated of negative modulation of a variety of other receptor types (Table 1). The concentration required for the negative modulation of many of these receptors is quite high, low micromolar concentrations rather than the nanomolar concentrations required for the positive modulation of GABAA receptors. This raises questions as to the likelihood that these interactions have physiological signiﬁcance. In addition to any putative role as pharmaceuticals, where their similarity of action to the benzodiazepines suggests their side effect proﬁle may be counterindicative, the neurosteroids are important endogenous modulators of physiological function. The fatigue associated with pregnancy, postmenstrual syndrome, and postnatal depression have all been linked to ﬂuctuations in neurosteroid concentrations (31). Given this, there will be considerable interest in applying functional molecular biological and genetic Table 1 Allosteric Modulation by a Variety of Neuroactive Steroids of Ionotropic and Metabotropic Receptors Allosteric modulation Receptor type GABAA

Positive 3a,5a-THP 3a,5b-THP 3a,5a-THDOC

nACh Glycine 5HT3

NMDA

AMPA KA Oxytocin Sigma type 1 (r1)

PS

17b-Estradiol progesterone DHEA-S

Negative PS DHEA-S Progesterone 3a,5a-THP Progesterone PS Estradiol (17a and b) Progesterone Testosterone 3a,5a-THP 17b-Estradiol Pregnanolone-S Pregnanolone hemisuccinate PS PS Progesterone PS

Abbreviations: THP, tetrahydroprogesterone; PS, pregnenolone sulfate; DHEA-S, dehydroepiandrosterone sulfate; THDOC, tetrahydrodeoxycorticosterone. Source: From Ref. 31.

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techniques to carefully dissect the complex pathways neurosteroids affect and to analyze the details of their interaction with the various receptor targets so that more selective pharmaceuticals can be designed to take advantage of these therapeutic routes. Neurodegeneration Galantamine acts as a positive allosteric modulator of neuronal nACh receptors. It increases the probability of channel openings, enhancing the apparent potency, but not the efﬁcacy, of nicotinic agonists, in activating various nACh receptor subtypes (32,33). Allosteric modulation of nACh receptors is not common for all cholinesterase inhibitors, for example, it is shown by neither donepezil nor rivastigmine (33). Although details of the allosteric mechanism await clariﬁcation, all possess a cationic nitrogen (at physiological pH) that is located at a ﬁxed distance from a phenolic group (32,34). By positively modulating presynaptic nACh receptors, galantamine facilitates GABAergic and glutamatergic neurotransmission. It is only recently that the allosteric action of galantamine at nACh receptors has been associated with its therapeutic effectiveness in the treatment of Alzheimer’s disease (35,36). Alzheimer’s disease is a multifactorial disorder but it has been suggested that cognitive impairments in Alzheimer’s disease are the result of modiﬁcation of cholinergic modulation of glutamatergic and GABAergic neurotransmission, especially in the hippocampus and frontal cortex (37,38). Developments in this ﬁeld may lead to improved therapies for other degenerative neurological pathologies, such as Parkinson’s and Huntington’s diseases. Endogenous Allosteric Modulation Zinc (Zn2þ) is now well established as an endogenous allosteric modulator at postsynaptic receptors, including all the glutamate receptors (NMDA, AMPA, KA), the adenosine triphosphate-activated purinoceptors P2X family of receptors, the GABAA receptors (39), and the glycine receptor (40). Zinc modulation is highly dependent on subunit composition (41), and whether the modulation is positive or negative may depend on the zinc concentration (40). GABAA receptor subtypes abc (the overwhelming majority) are relatively insensitive to Zn2þ whereas the ab, abd, and abe subtypes are highly sensitive to inhibition by Zn2þ at a concentration in the low micromolar range (41). This has led to the suggestion that the physiological role of zinc is not to modulate the postsynaptic GABAA receptors responsible for neuronal inhibition, but to regulate the tonic inhibition provided by the extrasynaptic GABAA receptors (41). One exception to this may be in individuals suffering absence epilepsy. Studies have revealed that these individuals express a mutated form of the c2 subunit of the GABAA receptor, which

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renders it insensitive to benzodiazepines (42–45). It has been shown further that this mutation not only renders the receptor insensitive to benzodiazepines but makes it far more sensitive to inhibition by Zn2þ (46). It was suggested that the altered inhibition of the receptor by zinc may lead to the abnormal spike-and-wave discharges characteristic of absence epilepsy. If this turns out to be correct, then a novel therapeutic strategy for absence epilepsy may involve an antagonist at the zinc binding site to prevent these abnormal neuronal discharges. If the targeting were to involve the mutated c2 subunit, then the action should be highly selective, thus minimizing side effects. In addition to zinc and the neurosteroids (see preceding text), other endogenous allosteric modulators include sodium, calcium, l-amino acids, glycine, amidated lipids (e.g., oleamide), peptides (e.g., 5-hydroxytryptamine moduline), and arachadonic acid. It has even been shown that classic neurotransmitters, 5-HT and histamine, modulate the nACh and NMDA receptors (3). Thus, while synthetic pharmaceuticals that act at allosteric sites are attractive therapeutic modulators of ionotropic receptors, it is clear that many of these receptors are allosterically modulated under normal physiological conditions by endogenous substances. This undoubtedly adds to the complexity that needs to be unraveled before a truly comprehensive picture of synaptic transmission mediated by ionotropic receptors emerges. However, it does suggest that there may be alternative, more subtle means available to modulate a particular neurotransmission process which avoids the need to target the receptor directly. ALLOSTERIC MODULATION OF METABOTROPIC RECEPTORS Metabotropic receptors, otherwise known as G-protein coupled receptors, represent the largest group of therapeutic targets. Most ligands act at the orthosteric site as agonists, competitive antagonists, or inverse agonists (to inhibit constitutive activity) (5). Individual metabotropic receptors can demonstrate enormous variation in effect through a number of mechanisms, including post-transcriptional mechanisms (splice variation, exon skipping, intron retention), receptor dimerization, multiple signalling pathways (different receptors activating the same G-protein, one receptor activating more than one G-protein, and one G-protein activating different signalling pathways, through different second messenger enzymes and ion channels), and G-protein subunit exchange (47). To add to this complexity, it is now known that metabotropic receptors may be regulated by endogenous allosteric mechanisms (5). Endogenous Allosteric Modulation of Metabotropic Receptors Sodium ions interact with a highly conserved aspartate residue in the cytoplasmic region of transmembrane segment 2 (TM2) of a number of

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metabotropic receptors, including adrenoceptor 2A (a2A), dopamine receptors (D2,4), adenosine receptors (A1, A2a, A3), and neurotensin receptors (NTS1) (48–52). It is suggested that Naþ binding to the negatively charged aspartate changes the receptor conformation so as to decrease agonist binding afﬁnity. This, in turn, inhibits G-protein activation. The more recent studies indicate that additional residues in the cytoplasmic regions of TM1, TM3, and TM7 may also be binding sites for Naþ (52,53). As with the ionotropic receptors (see preceding text), zinc (Zn2þ) allosterically modulates metabotropic receptors, including dopamine (D1,2) tachykinin (NK3), melanocortin (MC1), and adrenoceptor (b2) receptors (51,54–59). Unlike sodium, zinc is stored in synaptic vesicles and coreleased, in a calcium-dependent manner, with neurotransmitters, reaching synaptic concentrations of 300 mM (60). Thus, while Naþ provides a tonic inhibition to a wide variety of metabotropic receptors, Zn2þ can produce a spectrum of physiologically relevant effects. Allosteric sites for Zn2þ have been identiﬁed on intracellular loops linking transmembrane domains (b2) as well as at cytoplasmic regions of transmembrane domains (MC1, NK3). The calcium-sensing receptor is potentiated by L-amino acids through binding sites located in the extracellalar N-terminal region (61,62). Calcium itself potentiates GABAB heterodimeric receptors, through binding at a site proximal to the GABAB1 orthosteric site (63). Oleic acid and oleamide potentiate 5-HT2/7 (10,64) receptors and 5-HT-moduline inhibits 5-HT1B/1D receptors (65,66). Oleamide has been associated with the induction of sleep, and therefore manipulation of the 5-HT2/7 receptors through the oleamide binding site may offer novel therapeutics to overcome insomnia that would be free of the side effects associated with benzodiazepines. The 5-HT1B/1D receptors regulate the release of 5-HT, and thus inhibiting these receptors would lead to an increase in serotonergic signalling. The 5-HT moduline binding site may therefore represent an opportunity for novel therapeutic manipulation of serotonergic signalling in the treatment of anxiety and depression. Synthetic Allosteric Modulators of Metabotropic Receptors The metabotropic receptors do not share an overall amino acid sequence identity. There exists a number of distinct topologies with N- and C-terminal regions of varying size and complexity, but all possess seven a-helical transmembrane domains. All, when activated by ligand binding, activate an associated G-protein that in turn initiates various intracellular signalling cascades and/or activates or inhibits ion channel function (47). The metabotropic receptor family is normally divided into three ‘‘superfamilies,’’ A, B, and C, based on amino acid sequence homology (Table 2). The endogenous agonists of the family A (‘‘rhodopsin-like’’) metabotropic receptors bind to a crevice formed by transmembrane domains 3, 5, 6, and 7 (e.g., rhodopsin, adrenoceptors) or occasionally to extracellular

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Table 2 Examples of Metabotropic Receptor Families, Named After the Endogenous Agonist, in the Three Different Superfamilies Family A Light mACh Noradrenaline Dopamine Serotonin Histamine Adenosine Somatostatin Opiod Melanocortin

Tachykinins Neurotensin Bomesin Endothelin Cholecystokinin

GPH

Family B

Family C

Secretin VIP PACAP Glucagons Calcitonin PTH

Glutamate GABAB Calcium Sucrose Umani

Abbreviations: GPH, glycoprotein hormone; mACh, muscarinic acetylcholine; VIP, vasoactive intestinal peptide; GABAB, c-aminobutyric acid type B; PACAP, pituitary adenylate cyclaseactivating peptide; PTH, parathyroid hormone.

regions of the receptor (e.g., glycoprotein hormone receptors). Family B metabotropic receptors bind peptides to both extracellular and transmembrane regions. Family C metabotropic receptors (e.g., glutamate, GABA) bind the endogenous agonists exclusively in the large N-terminal domain (5). Many synthetic ligands for both families A and C metabotropic receptors have been developed that act to allosterically modulate these receptors. Allosteric Modulators of Family A Metabotropic Receptors Allosteric modulators have been identiﬁed for many family A metabotropic receptors, including a1A and a2A adrenoceptors, adenosine receptors, chemokine receptors, dopamine receptors, serotonin receptors, and muscarinic receptors (5). The ﬁrst mACh allosteric modulators (e.g., gallamine) were shown to inhibit cardiac mACh receptors. A wide range of structurally diverse compounds have since been shown to act allosterically. Although the most sensitive mACh receptor subtype to allosteric modulation is M2, no clearly subtype-speciﬁc allosteric modulator has been identiﬁed, probably due to the high degree of amino acid sequence identity between the ﬁve mACh subtypes. Most of the allosteric modulators, including the negative modulator gallamine and the positive modulator alcuronium, bind through a ‘‘common allosteric site’’ on the extracellular side of the orthosteric site. The extracellular loops linking the transmembrane domains (especially the second and third) are crucial to this site. More recently, speciﬁc amino acid residues in mACh-M2 receptors that are critical, but not exclusive, for alkane-bisammonium and caracurine V allosteric ligand binding have been identiﬁed (Tyr177 in the second extracellular loop and Thr423 in TM7).

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Some allosteric ligands (e.g., KT5720 and various WIN62,577 analogs) do not act through the common allosteric site demonstrating that there are multiple allosteric sites on the muscarinic receptors. Allosteric Modulators of Family C Metabotropic Receptors A large number of potent allosteric inhibitors and potentiators of metabotropic glutamate receptors have been identiﬁed which are highly selective for speciﬁc subtypes (Table 3) (5). Additionally, potentiators and inhibitors have been identiﬁed for the calcium-sensing receptor and three allosteric potentiators have been identiﬁed for the heterodimeric GABAB1/2 receptor. The binding sites of allosteric inhibitors for glutamate receptors (CPCCOEt, BAY36–7620, EM-TBPC, R 214127, and MPEP) and allosteric Table 3 Allosteric Modulators of Family C Metabotropic Receptors Receptor

Positive

Negative

mGluR1

Ro 01–6128 Ro 67–7476

mGluR2 mGluR4

LY 487379 MPEP PHCCC DFB NPS R-467 NPS R-568 GS 39783 CGP 7930 CGP 13501

CPCCOEt PHCCC BAY 36–7620 EM-TBPC R 214127 GSK compounds NPS 2390 Ro 64–5229

mGluR5 Calcium receptor GABAB1/2

MPEP Calhex 231

Abbreviations: CPCCOEt, 7-(hydroxyimino)cyclopropan[b]chromen-1a-carboxylic acid ethyl ester; PHCCC, N-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide; BAY36– 7620, (3aS,6aS)-6a-naphthalen-2-ylmethyl-5-methyliden; EM-TBPC, 1-ethyl-2-methyl-6-oxo4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydro-pyrimidine-5-carbonitrile; R 214127, [(3)H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone; NPS 2390, 2-quinoxaline-carboxamide-N-adamantan-1-yl; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; Calhex 231, (1S,2S,10 R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclohexane; Ro 01–6128, diphenyl-acetyl-carbamic acid ethyl ester; Ro 67–7476, (S)-2-(4-ﬂuoro-phenyl)-1(toluene-4-sulfonyl)-pyrrolidine; LY 487379, N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-triﬂuoroethylsulfonyl)pyrid-3-ylmethylamine; GS 39783, N,N0 -dicyclopentyl-2-(methylthio)-5-nitro-4,6pyrimidinediamine; CGP7930, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol; CGP 13501, aldehyde analog of CGP7930. Source: From Ref. 5.

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potentiators for glutamate receptors (Ro 01–6128, Ro 67–7476, LY 487379) and allosteric potentiators for calcium-sensing receptors (NPS R-467 and NPS R-568) reside exclusively within the 7TM domains, removed from the orthosteric site located in the N-terminal domain (5). The speciﬁc residues responsible for the subtype selectivity have been identiﬁed and are widely dispersed throughout the transmembrane regions. It has been concluded that all the allosteric modulators bind in a crevice deﬁned by the four transmembrane domains TM3, TM5, TM6, and TM7 (5). A common allosteric site in the transmembrane domain of the family C GPCR, which mediates both allosteric potentiation and inhibition, mirrors the extracellular common allosteric site in the mACh receptors, and may underlie the structural differences between allosteric inhibitors and potentiators of the family C receptors. Allosteric inhibitors of mGluR1 or mGluR5 (PHCCC, SIB1893, and MPEP) are weak allosteric potentiators of mGluR4 receptors. A series of benzaldazine analogs (e.g., DFB in Table 3) exhibit at mGluR5 receptors a range of activity from allosteric potentiation to allosteric inhibition to neutral cooperativity. The number of allosteric ligands identiﬁed for mACh receptors and mGluRs compared to other GPCRs probably reﬂects an intensive search for allosteric modulators for these receptors, given their importance in a range of cardiovascular and neurological pathologies. For example, modulation of mGluR1 may well be a novel means of controlling hyperalgesia and also excitotoxicity, while mGluR5 inhibition may reduce anxiety and assist in the treatment of drug dependence and motor dysfunction in Parkinson’s disease (67). CONCLUSIONS Despite the obvious potential therapeutic beneﬁts, in terms of efﬁcacy and safety, of the prototypic allosteric modulators, the benzodiazepines, the search for compounds which would provide the clinical proﬁle without the side effects was unsuccessful. Partly as a consequence, with the emerging knowledge of receptor structure, the development of combinatorial chemistry, and the need to introduce suitably high throughput screening methods, the wealth of new compounds to test tended to be of the agonist/antagonist type. The driving philosophy appears to have been a belief that, by using the improving knowledge of receptor structure, compounds more selectively targeted to speciﬁc receptor subtypes would be designed which, because of their selective binding, would provide efﬁcacy and fewer side effects. It appears that this effort has not been rewarding in terms of clinically useful new therapeutics, but it has driven forward our understanding of the regulation of receptor function. Our vastly improved knowledge of receptor structure and the relationship between structure and function has derived from successes in

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deﬁning ever more precisely receptor structure, using both imaging and molecular biological techniques, and in combining functional analysis with molecular biological and genetic techniques. These developments have led to a resurgence of interest in understanding how allosteric modulation of receptor function comes about and what a fundamental aspect of the normal physiological regulation of ionic signalling it is. Metabotropic receptor regulation, in particular, now seems to be almost bewilderingly complex, with genetic, structural, allosteric, and physiological mechanisms all operating simultaneously. However, with the techniques available it should be possible to develop strategies that utilize unique allosteric binding sites to subtly alter selective receptor function in ways that will provide therapeutic beneﬁt without unwanted toxicology. REFERENCES 1. Muir KW, Lees KR. Clinical experience with excitatory amino acid antagonist drugs. Stroke 1995; 26:503–513. 2. Mo¨hler H, Fritschy JM, Rudolph U. A new benzodiazepine pharmacology. J Pharmacol Exp Ther 2002; 300:2–8. 3. Christopoulos A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat Rev Drug Discov 2002; 1:198–210. 4. Birdsall NJM, Farries T, Gharagozloo P, Kobayashi S, Lazareno S, Sugimoto M. Subtype-selective positive cooperative interactions between brucine analogs and acetylcholine at muscarinic receptors: functional studies. Mol Pharmacol 1999; 55:778–786. 5. Jensen AA, Spalding TA. Allosteric modulation of G-protein coupled receptors. Eur J Pharm Sci 2004; 21:407–420. 6. Tucek S, Musilkova J, Nedoma J, Proska J, Shelkovnikov SW, Vorlicek J. Positive cooperativity in the binding of alcuronium and N-methylscopolamine to muscarinic acetylcholine receptors. Mol Pharmacol 1990; 38:674–680. 7. Christopoulos A, Mitchelson F. Use of a spreadsheet to quantitate the equilibrium binding of an allosteric modulator. Eur J Pharmacol 1998; 355: 103–111. 8. Kostenis E, Mohr K. Composite action of allosteric modulators on ligand binding. Trends Pharmacol Sci 1996; 17:443–444. 9. Litschig S, Gasparini F, Rueegg D, et al. CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signalling without affecting glutamate binding. Mol Pharmacol 1999; 55:453–461. 10. Thomas EA, Carson MJ, Neal MJ, Sutcliffe JG. Unique allosteric regulation of 5-hydroxytryptamine receptor-mediated signal transduction by oleamide. Proc Natl Acad Sci USA 1997; 94:14115–14119. 11. Hedlund PB, Carson MJ, Sutcliffe JG, Thomas EA. Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors. Biochem Pharmacol 1999; 58:1807–1813. 12. Korpi ES, Gru¨nder G, Lu¨ddens H. Drug interactions at GABAA receptors. Prog Neurobiol 2002; 67:113–159.

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13. Atack JR, Hutson PH, Collinson N, et al. Anxiogenic properties of an inverse agonist selective for a3 subunit-containing GABAA receptors. Br J Pharmacol 2005; 144:357–366. 14. Wafford KA. GABAA receptor subtypes: any clues to the mechanism of benzodiazepine dependence? Curr Opin Pharmacol 2005; 5:47–52. 15. Costa E. From GABAA receptor diversity emerges a uniﬁed vision of GABAergic inhibition. Ann Rev Pharmacol Toxicol 1998; 38:321–350. 16. Sieghart W, Sperk G. Subunit composition distribution function of GABAA receptor subtypes. Curr Top Med Chem 2002; 2:795–816. 17. Casula MA, Bromidge FA, Pillai GV, et al. Identiﬁcation of amino acid residues responsible for the a5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABAA receptor. J Neurochem 2001; 77:445–451. 18. Downie DL, Franks NP, Lieb WR. Effects of thiopental and its optical isomers on nicotinic acetylcholine receptors. Anesthesiology 2000; 93:774–783. 19. Violet JM, Downie DL, Nakisa RC, Lieb WR, Franks NP. Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. Anesthesiology 1997; 86:866–874. 20. Downie DL, Vicente-Agullo F, Campos-Caro A, Bushell TJ, Lieb WR, Franks NP. Determinants of the anesthetic sensitivity of neuronal nicotinic acetylcholine receptors. J Biol Chem 2002; 277:10367–10373. 21. Parker RM, Bentley KR, Barnes NM. Allosteric modulation of 5-HT3 receptors: focus on alcohols and anaesthetic agents. Trends Pharmacol Sci 1996; 17:95–99. 22. Zimmerman SA, Jones MV, Harrison NL. Potentiation of gamma-aminobutyric acidA receptor Cl current correlates with in vivo anesthetic potency. J Pharmacol Exp Ther 1994; 270:987–991. 23. Belelli D, Pistis M, Peters JA, Lambert JJ. The interaction of general anaesthetics and neurosteroids with GABAA and glycine receptors. Neurochem Int 1999; 34:447–452. 24. Mihic SJ, Ye Q, Wick MJ, et al. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature 1997; 389:385–389. 25. Daniels S, Roberts RJ. Post-synaptic inhibitory mechanisms of anaesthesia; glycine receptors. Toxicol Lett 1998; 100–101:71–76. 26. de Sousa SL, Dickinson R, Lieb WR, Franks NP. Contrasting synaptic actions of the inhalational general anesthetics isoﬂurane and xenon. Anesthesiology 2000; 92:1055–1066. 27. Yamakura T, Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels: comparison with isoﬂurane and ethanol. Anesthesiology 2000; 93:1095–1101. 28. Reynolds DS, Rosahl TW, Cirone J, et al. Sedation and anesthesia mediated by distinct GABAA receptor isoforms. J Neurosci 2003; 23:8608–8617. 29. Wang MY, Rampil IJ, Kendig JJ. Ethanol directly depresses AMPA and NMDA glutamate currents in spinal cord motor neurons independent of actions on GABA(A) or glycine receptors. J Pharmacol Exp Ther 1999; 290:362–367. 30. Dinsel A, Fo¨hr KJ, Georgieff M, Beyer C, Bulling A, Weigt HU. Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones. Br J Anaesth 2005; 94:479–485.

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48. Horstman DA, Brandon S, Wilson AL, Guyer CA, Cragoe EJ Jr, Limbird LE. An aspartate conserved among G-protein receptors confers allosteric regulation of a2-adrenergic receptors by sodium. J Biol Chem 1990; 265:21590–21595. 49. Neve KA, Cox BA, Henningsen RA, Spanoyannis A, Neve RL. Pivotal role for aspartate-80 in the regulation of dopamine D2 receptor afﬁnity for drugs and inhibition of adenylyl cyclase. Mol Pharmacol 1991; 39:733–739. 50. Martin S, Botto JM, Vincent JP, Mazella J. Pivotal role of an aspartate residue in sodium sensitivity and coupling to G proteins of neurotensin receptors. Mol Pharmacol 1999; 55:210–215. 51. Schetz JA, Sibley DR. The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation. J Pharmacol Exp Ther 2001; 296:359–363. 52. Gao ZG, Kim SK, Gross AS, Chen A, Blaustein JB, Jacobson KA. Identiﬁcation of essential residues involved in the allosteric modulation of the human A3 adenosine receptor. Mol Pharmacol 2003; 63:1021–1031. 53. Neve KA, Cumbay MG, Thompson KR, et al. Modelling and mutational analysis of a putative sodium-binding pocket on the dopamine D2 receptor. Molecular Pharmacology 2001; 60:373–381. 54. Schetz JA, Sibley DR. Zinc allosterically modulates antagonist binding to cloned D1 and D2 dopamine receptors. J Neurochem 1997; 68:1990–1997. 55. Rosenkilde MM, Lubicello M, Holst B, Schwartz TW. Natural agonist enhancing bis-His zinc-site in transmembrane segment V of the tachykinin NK3 receptor. FEBS Lett 1998; 439:35–40. 56. Schetz JA, Chu A, Sibley DR. Zinc modulates antagonist interactions with the D2-like dopamine receptors through distinct molecular mechanisms. J Pharmacol Exp Ther 1999; 289:956–964. 57. Holst B, Elling CE, Schwartz TW. Metal-ion mediated agonism and agonistenhancement in the melanocortin MC1 and MC4 receptors. J Biol Chem 2002; 277:47662–47670. 58. Swaminath G, Lee TW, Kobilka B. Identiﬁcation of an allosteric binding site for Zn2þ on the b2 adrenergic receptor. J Biol Chem 2002; 278:352–356. 59. Swaminath G, Steenhuis J, Kobilka B, Lee TW. Allosteric modulation of b2adrenergic receptor by Zn2þ. Mol Pharmacol 2002; 61:65–72. 60. Frederickson CJ. Neurobiology of zinc and zinc-containing neurons. Int Rev Neurobiol 1989; 31:145–238. 61. Conigrave AD, Quinn SJ, Brown EM. L-amino acid sensing by the extracellular Ca2þ-sensing receptor. Proc Natl Acad Sci USA 2000; 97:4814–4819. 62. Zhang Z, Qiu W, Quinn SJ, Conigrave AD, Brown EM, Bai M. Three adjacent serines in the extracellular domains of the CaR are required for L-amino acid-mediated potentiation of receptor function. J Biol Chem 2002; 277: 33727–33735. 63. Pre´zeau L, Galvez T, Kaupmann K, et al. A single residue in GABA-B receptor Type 1 is responsible for the Ca-sensing property of the GABA-B receptor heteromer. Neuropharmacology 1999; 38:A30–A36. 64. Alberts GL, Chio CL, Im WB. Allosteric modulation of the human 5-HT7A receptor by lipidic amphipathic compounds. Mol Pharmacol 2001; 60:1349–1355.

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65. Fillion G, Rousselle JC, Massot O, Zifa E, Fillion MP, Prudhomme N. A new peptide, 5-HT-moduline, isolated and puriﬁed from mammalian brain speciﬁcally interacts with 5-HT1B/1D receptors. Behav Brain Res 1996; 73:313–317. 66. Massot O, Rousselle JC, Fillion MP, et al. 5-Hydroxytryptamine-moduline, a new endogenous cerebral peptide, controls the serotonergic activity via its speciﬁc interaction with 5-hydroxytryptamine1B/1D receptors. Mol Pharmacol 1996; 50:752–762. 67. Gasparini F, Kuhn R, Pin J-P. Allosteric modulators of group I metabotropic glutamate receptors: novel subtype-selective ligands and their therapeutic perspectives. Curr Opin Pharmacol 2002; 2:43–49.

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2 Characteristics of Allosterism in Drug Action Terry P. Kenakin Assay Development, GlaxoSmithKline Research and Development, Research Triangle Park, North Carolina, U.S.A.

GLOBAL PROTEIN PERTURBATION G-protein–coupled receptors (GPCRs) are natural allosteric proteins. They allow small molecules such as neurotransmitters and hormones to cause changes in protein–protein interactions in the form of the binding of receptors and G-proteins. Since agonists access receptors from the extracellular side of the cell membrane to initiate changes in the interaction of the receptor and G-proteins on the intracellular side of the membrane, the effect must be transduced through the protein. The interaction of the receptor with the agonist causes a bias in the conformation of the receptor and this new shape of the receptor protein has different properties (in this case, activation of G-proteins). There is a fundamental difference between conventional ideas of steric hindrance of molecular interaction on proteins versus allosteric perturbation. In the former case, two molecules compete for a single binding site on the protein; blockade of the binding of one comes about by a steric hindrance of the binding of the other, i.e., occupation of the single binding site by one ligand precludes binding of the other. In contrast, for allosteric interactions, both ligands bind to separate sites on the receptor and interactions occur through the protein. Since the receptor response is a change in tertiary conformation, there are no rules for how extensive the change brought about by the allosteric can be. There are cases where 19

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the binding of small druglike molecules cause very striking changes in the interactions of very large proteins. For example, the infection of cells with HIV-1 is caused by the interaction of the chemokine receptor type C 5 (CCR5; molecular weight 50,000), the membrane protein CD4, and the viral coat protein gp120 (molecular weight ¼ 120,000) (1–6). Point mutation studies have shown that relatively large areas of the CCR5 receptor (speciﬁcally all four extracellular domains) are involved in the fusion process (7–13). Mutational studies with gp120 also suggest that multiple regions of this protein are involved with CCR5-mediated HIV infection (12–16). It has also been shown that blockade of HIV infection is not amenable to single point mutations of the CCR5 molecule (17). In spite of the size of the proteins involved and the fact that large areas of these proteins interact with each other to initiate HIV fusion, small drug-like molecules (molecular weight ¼ 500–600) block this process with nanomolar potencies (18–21). These remarkable effects raise the question, how can such small molecules cause such large global changes in protein conformation? A useful theoretical approach to this question is found in protein ensemble theory. While it is convenient to describe receptor conformations in terms of one or two states (i.e., active and inactive with respect to G-protein activation), it probably is simplistic to do so. It has long been proposed (22) that proteins exist in a number of energetically different states (23–26) and that they sample those states continually in conformational space (27,28). This is not a new idea, having been proposed for proteins over 40 years ago: ‘‘ . . . a protein cannot be said to have ‘a’ secondary structure but exists mainly as a group of structures not too different from one another in free energy . . . In fact, the molecule must be conceived as trying every possible structure . . . ’’ (29). These concepts have been developed for enzymes (30–32) and have also been applied to receptors (33–37). Within this context, a ligand has a range of afﬁnities for this collection of states (ensemble) and will bias the makeup of the ensemble according to this spectrum of afﬁnities. The conformations for which the ligand has the highest afﬁnity will be enriched selectively at the expense of other conformations in the ensemble, a process referred to as ‘‘conformational selection’’ (38). Within the framework of this molecular mechanism, small molecules can produce complete changes in the receptor protein conformation through selective binding. A useful way to visualize allosterically induced global differences in receptor conformation is with a model ﬁrst described by Ehlert (39); the differences are deﬁned by a ‘‘cooperativity constant’’ denoted a. Thus, if the afﬁnity of a receptor for a ligand A is Ka, the afﬁnity modiﬁed by an allosteric ligand [B] is denoted as aKa (see following scheme). The magnitude of the parameter a deﬁnes the overall effect of the allosteric ligand on the receptor. Thus, if a < 1, the allosteric effect is antagonistic, i.e., for a ligand [B] with a ¼ 0.1, the afﬁnity of the receptor is 1/10

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Characteristics of Allosterism in Drug Action

αKa

A + RB Kb

21

ARB αKb

Ka A +

R + B

AR + B

for ligand [A] when [B] is bound. Similarly, if a > 1, the afﬁnity of the receptor is enhanced for the ligand. The global nature of the changes brought about by an allosteric ligand to a receptor is illustrated by the effects of the ligand eburnamonine on muscarinic m2 receptors. Figure 1 shows the pEC50 values (log of the molar 7.0 6.5 6.0

APE Arecoline Acetylcholine Carbachol Methylfurmethide Oxotremorine Pilocarpine

pEC50

5.5 5.0 4.5 4.0 3.5 3.0 Native Receptor

+ Eburnamonine

Figure 1 Potency values (pEC50, log molar concentration producing half the maximal response) of muscarinic agonists for m2 receptors in the absence and presence of saturating concentrations of the allosteric modulator eburnamonine. It can be seen that the modulator completely changes the rank order of potency of these agonists, proﬁles indicative of completely different receptors. Rank order of potency ratios on native receptor: APE > arecoline > oxotremorine > methylfurmethide > pilocarpine > Ach > carbachol. In the presence of eburnamonine: arecoline > APE > methylfurmethide > oxotremorine > carbachol > Ach > pilocarpine. Abbreviation: APE, arecaidine propargyl ester. Source: From Ref. 40.

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Table 1 Cooperativity Constants for Muscarinic Agonists Produced by Eburnamonine (Muscarinic m2 Receptors) Agonist Acetylcholine Arecoline Methylfurmethide Pilocarpine Oxotremorine Carbachol Arecaidine propargyl ester

a 0.32 15 0.64 0.04 0.24 0.6 0.88

Source: From Ref. 40.

concentration producing 50% maximal response) of seven muscarinic receptor agonists in the absence and presence of a saturating concentration of eburnamonine (40). It can be seen that, not only does this allosteric modulator change the potencies of the agonists, but it also completely changes the rank order of their potency; this latter parameter is a standard pharmacological identiﬁer of receptor types. These data indicate that eburnamonine essentially produces a receptor that is intrinsically different from the native m2 receptor. These data also illustrate an important aspect of allosteric modiﬁcation of receptors, namely that the changes are very much probe speciﬁc, i.e., every allosteric modulator has a unique value of a for every receptor probe. The values of a for eburnamonine and the muscarinic agonists are given in Table 1. Allosteric modulators also can have strikingly different effects on a single receptor probe as in the case of [3H]-N-methylscopolamine binding to muscarinic receptors. While the allosteric ligand alcuronium enhances the afﬁnity for the same probe (a ¼ 10), the allosteric ligand gallamine is an allosteric antagonist (a ¼ 0.067) (41). PRACTICAL ASPECTS OF ALLOSTERIC PROBE DEPENDENCE There are experimental and therapeutic implications of allosteric probe dependence. For example, for two radioligand probes of the muscarinic m2 receptor, [3H] methyl quinucidyl benzylate and [3H] atropine, completely opposite effects are seen with the allosteric ligand alcuronium. Speciﬁcally, alcuronium completely blocks the binding of [3H] methyl quinucidyl benzylate (QNB) but increases the binding of [3H] atropine by 250% on muscarinic m2 receptors (42). Such probe dependence underscores the notion that the endogenous natural agonist should be employed whenever possible in functional experiments with allosteric ligands. For example, if the application is potentiation of cholinergic transmission (as in Alzheimer’s disease—vide infra), and the cholinergic test agonist is arecoline, a 15-fold potentiation

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of muscarinic m2 receptor response is obtained with the allosteric modulator eburnamonine. However, this potentiation is not observed for the natural agonist acetylcholine; in fact, a threefold antagonism of cholinergic results with this modulator (40). Therefore, the activity predicted by the surrogate agonist arecoline is completely misleading and would not lead to physiologically relevant potentiation of acetylcholine function. Though preferable, it is not always possible to do primary testing with the endogenous receptor probe. For example, while screening with live HIV is theoretically preferred when screening for HIV co-receptor antagonists, safety and containment issues make this impractical. For safety reasons, radioligand binding assays with 125I-chemokine on CCR5 receptors are used as surrogate screening assays. Therefore, activity detected with one probe (i.e.,125I-MIP-1a) is applied to alteration of another probe (namely gp120 viral coat protein of HIV). Follow-up studies with the physiologically relevant probe, in this case HIV, indicate instances where dissimulations occur. However, antagonists that retain potency as blockers of 125I-MIP-1a binding can be shown to depart from HIV activity by factors of 2000 (19). The damaging aspect of this is not in cases where relevant activity detected in the screen does not extend to activity with HIV but rather in the unknown number of cases where a physiologically relevant initial test (HIV) would have detected activity missed in the surrogate screen. Possible shortcomings in surrogate screening also can affect the type of assay used for detection. For example, although there are many allosteric effects resulting in changes in probe afﬁnity (and thus sensitive to receptor binding), there also are instances where allosterism affects only receptor function and not receptor occupancy. For example, the ligand 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ester (CPCCOEt) does not alter glutamate binding but is a potent noncompetitive antagonist of glutamate receptor response (43). Similarly, responses through allosteric activation of muscarinic m2 receptors mediated by the allosteric agonist alcuronium are impervious to blockade by the classical muscarinic antagonist quinucidyl benzylate (44). Clearly, a binding assay with [3H]-QNB as the receptor probe would not have detected this agonist activity. Allosteric probe dependence can be advantageous for the preservation of physiological function with concomitant pathologically based antagonism; this may be relevant in the blockade of X4 strain HIV infection through blockade of the chemokine receptor CXCR4. The CXCR4 receptor has been shown to be a mediator of HIV X4 infection (45); however, loss of CXCR4 receptor function can be deleterious to normal physiological function. For example, deletion of the genes known to mediate expression of the CXCR4 receptor or the natural agonist for CXCR4 (stromal cell derived factor 1-a, SDF-1a) is lethal and leads to developmental defects in the cerebellum, heart, and gastrointestinal tract as well as hematopoiesis (46–48). These data indicate that antagonism of CXCR4-mediated HIV

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infection should be accompanied by retention of normal chemokine (i.e., mediated by SDF-1a) activation of CXCR4. Interestingly, ligand-mediated divergence of physiological activity and mediation of HIV entry has been reported for CXCR4. For example, peptide-agonists for CXCR4 RSVM and ASLW are not blocked by the CXCR4 antagonist AMD3100, which itself is a potent antagonist of HIV entry (49). Dissociation between HIV and chemokine activity also is observed with peptide fragments of SDF-1a. Speciﬁcally, while the ratio of inhibitory concentration (IC50) values for the blockade of HIV entry and chemokine function for the peptide fragment 1–13 is 0.25, the same ratio for the fragment L-5H is 60-fold greater (IC50 function/IC50-HIV ¼ 15) (50). These data suggest the possibility that, in the future, molecules can be found that block HIV entry but not interfere with CXCR4-mediated chemokine function. PROBE-DEPENDENT ANTAGONISM For orthosteric competitive antagonists, a receptor blocked by two different antagonists is equivalent, i.e., agonist-binding is precluded. In contrast, for allosteric antagonists, there is texture in antagonism as deﬁned by the magnitude of the cooperativity constant a. Thus, two allosterically blocked receptors may apparently be equivalent from one viewpoint but completely unique with respect to other receptor functions as measured by other receptor probes, i.e., different probes of the receptor protein may show a greater texture in the differences between the antagonist-bound receptors. For example, the b-adrenoceptor antagonist alprenolol does not produce positive or negative signaling of the b-adrenoceptor (in terms of G-protein signaling it is a neutral antagonist). However, a covalent ﬂourescent label at cysteine 265, a region sensitive to receptor conformational change, indicates that the conformations sampled by this receptor are altered when alprenolol is bound, i.e., a different probe of the receptor shows that an active change in the receptor occurs with antagonist binding (51). Similar differences in antagonism by molecules have been revealed for enzymes as well. For example, while most inhibitors of human renin access the b-strand binding enzyme conformation, novel piperidine inhibitors have been shown to stabilize a completely different conformation (52). These two chemical series produce the same therapeutic endpoint (i.e., renin blockade) through stabilization of different protein conformations. The fact that CCR5 antagonism through allosterism brings a texture to antagonism may have direct therapeutic implications. One example of where this may be relevant is in resistance to therapy against HIV infection. Speciﬁcally, chronic use of CCR5 antagonists theoretically may lead to selective pressure on the virus to mutate to resistant strains. Thus, through mutation, the viral envelope proteins may evolve to forms that are able to fuse with the antagonist-bound receptor. However, if other allosteric ligands

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stabilize new conformations of CCR5 (through stabilization of different conformational ensembles) then the new allosterically induced conformation may block the resistant strain. Thus, sequential use of allosteric antagonists may be a viable means of avoiding long-term viral resistance. UNIQUE PROPERTIES OF ALLOSTERIC LIGANDS In addition to probe dependence, allosteric ligands possess certain other properties that can make them unique for drug therapy. Speciﬁcally, these properties are: 1. Allosteric ligands potentially can produce subsets of receptor behavior. 2. There are separate binding sites for allosteric ligands and receptor probes. 3. Preservation of physiological patterns can occur with allosteric modulation. 4. Saturability of allosteric effect enables possible dissociation of the intensity of effect from the duration of effect. It is worth considering each of these. Protein Ensembles and Subsets of Receptor Behavior The existence of different ensembles of receptor conformations and the association of those ensembles with various physiological functions opens the possibility of allosteric redirection of receptor conformation toward selected therapeutic pathways (35,36). This is in contradistinction to the conventional view of efﬁcacy as presented by Stephenson (53) in which a more linear progression of receptor behavior is assumed, i.e., receptor activation leads to desensitization, phosphorylation, and subsequent internalization. Recent data suggest that some of these behaviors may be initiated independently without the others through ligand-speciﬁc receptor active states (54). For example, although receptor internalization traditionally is linked to agonist-activation of receptors, the cholecystokinin (CCK) receptor antagonist D-Tyr-Gly-[(Nle28,31,D-Trp30) cholecystokinin-26-32]phenethyl ester, produces receptor internalization with no concomitant receptor activation (55). Similarly, antibodies can select for CCR5 receptor behaviors. Thus, the antibody MC-4 does not block chemokine binding or function or induce receptor internalization but it does block AOPRANTES-induced receptor internalization (56). Similarly, the antibody MC-1 blocks chemokine binding and does not produce agonism, but does induce receptor internalization (56). The mechanism for these effects most likely is allosteric global perturbation of the receptor through ligand conformational selection.

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Ligand selection of distinct receptor behaviors has the potential of leading to therapeutically useful activity. One area where this may be important is in the selective induction of CCR5 receptor internalization. In the case of CCR5, this would remove the target for HIV-1 to a point whereby no amount of viral mutation could overcome blocked CCR5, i.e., the receptor would not be present for binding of the virus to the cell membrane. Separate Binding Sites on the Receptor Interaction of molecules through receptor protein conformation implies separate binding loci on the receptor. Parenthetically, it should be noted that allosteric behavior may formally appear to be orthosteric (a single binding site shared by both the probe and allosteric molecule) in cases of very low values of the cooperativity constant, i.e., a ¼ 0.0001. Such low cooperativity values indicate that the binding of the allosteric modulator effectively precludes the binding of the probe. However, this is not evidence of commonality of binding sites as it could simply mean that the allosteric modulator produces a receptor conformation with a very low afﬁnity for the probe. From the point of view of ligand selectivity, separate binding sites on the receptor protein for allosteric modulators can be interpreted in alternate ways. On the one hand, it could be argued that separate modulator binding sites can be a mechanism for increased ligand selectivity for receptors, as in the case of muscarinic receptors. Here it is suggested that muscarinic receptor subtypes have evolved teleologically to recognize a common endogenous ligand, namely acetylcholine; thus it would not be expected that orthosteric ligands designed to sterically interfere with acetylcholine binding to this site would demonstrate receptor subtype selectivity. In contrast, other sites on the receptor that have evolved to generate the subtype differences in the muscarinic receptor and thus give the subtype character to the receptor may in fact offer speciﬁc binding sites for selective ligands (57). Figure 2 shows a diagram depicting the afﬁnity of the endogenous muscarinic agonist acetylcholine and three other agonists on four subtypes of muscarinic receptor. In addition, this ﬁgure also shows the afﬁnities of ﬁve allosteric modulators of muscarinic receptors. It can be seen that while the relative potency of acetylcholine and the other agonists is extremely uniform over the four receptor subtypes, a much greater variance in potencies is seen with the allosteric modulators, i.e., these latter molecules discern the muscarinic receptor subtypes. From a drug discovery point of view, this theoretically also offers a potential cornucopia of active binding sites for selective modulators, i.e., the entire surface of the receptor protein might be considered a potential source of ligand binding sites. On the other hand, allosteric modulators may access common allosteric binding sites on different receptors and thus be relatively nonselective.

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Characteristics of Allosterism in Drug Action

Agonists

27

Allosteric Modulators

6.5 6.0

Acetylcholine

pKA or pKB

5.5

Carbachol

5.0

Bethanechol

4.5

Alcuronium Strychnine Brucine

4.0

Vincamine Eburnamonine

3.5 3.0

m1

m2

m3

m4

m1

m2

m3

m4

Receptor Subtypes Figure 2 Potency (expressed as log molar concentration producing half maximal receptor occupancy) of agonists and antagonists on four subtypes of muscarinic receptor. Source: From Ref. 40.

For example, the ligand SCH-202676, in the concentration range of 0.1– 1.8 mM, blocks radioligand binding on j-, m-, d-opioid receptors, b-, a-adrenoceptors, D1, D2 dopamine receptors and m1, m2 muscarinic receptors (58). Preservation of Physiological Patterns Patterns of innervation, blood ﬂow, cellular receptor density, and efﬁciencies of receptor coupling lead to complex systems of physiological control in the brain and other organs. Failure of some of these systems can lead to disease states as in the case of Alzheimer’s disease, where it has been postulated that a reduction in cholinergic function results in cognitive and memory impairment (59). Whole-scale activation of cholinergic receptors in the brain would lead to nonspeciﬁc excitation, receptor desensitization, and downregulation. One clinical approach to reduce such nonspeciﬁcity is the application acetylcholinesterase antagonists; theoretically this should lead to somewhat selective augmentation of cholinergic function in synapses (60). However, this approach appears to have limited practical value (61–63). In this case it may be that cholinesterase inhibitors produce relatively nonselective increases in cholinergic function through both nicotinic and muscarinic receptors (62), whereas selective potentiation of nicotinic responses is required (63). A more selective approach has been proposed based on the allosteric potentiation of nicotinic receptor function (64,65).

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Theoretically, this approach would selectively augment function where it is needed in direct proportion to the pattern laid down by nicotinic receptor expression levels. Since allosteric modulators are not independent agonists for the receptor, it would be envisaged that little loss of selectivity or overstimulation would result (although the latter point is not an automatic conclusion). The key feature of allosteric potentiation of receptor effect is the association of the augmentation with the initial magnitude of response. Thus, for an allosteric potentiator with a ¼ 10, there is a 10-fold shift to the left of the dose–response curve for acetylcholine on the affected receptors. However, in the absence of cholinergic excitation, there would be no effect of the allosteric modulator. Theoretically, such input-associated control of response should reﬂect the natural texture of the system brought on by patterns of innervation and receptor expression. Dissociation Between Duration and Intensity of Effect Another important feature of allosteric receptor modulation is the fact that the effect is saturable, i.e., the allosteric site can be maximally occupied to produce a maximal effect. This is in contrast to orthosteric competitive systems where the effect of the antagonist theoretically can be inﬁnite. In this latter type of system, the concentration of the antagonist controls both the magnitude and duration of the effect, thereby associating these two parameters. For allosteric compounds, these parameters will not be associated once the allosteric site is maximally occupied. When this occurs, no further maximal response will be seen (this is controlled by the magnitude of the cooperativity constant a), but the duration of effect will increase. Under ideal circumstances, the receptor compartment can be saturated with the allosteric modulator to produce a long duration of a constant effect. THE DETECTION AND QUANTIFICATION OF ALLOSTERIC EFFECT In view of the unique properties of allosteric modulators, it is advantageous to differentiate allosteric from orthosteric receptor function. Depending upon the magnitude of the cooperativity constant of the modulator for the receptor-probe combination, allosteric effects may sometimes appear to be simple competitive interactions. However, there are two underlying themes associated with allosteric function that can be explored to elucidate mechanism. These are that allosteric effects are (i) saturable and (ii) probe dependent. The ﬁrst general property of allosterism can be used in some circumstances to detect and elucidate the allosteric nature of the receptor interaction. Figure 3 shows the effects of an allosteric antagonist of a ¼ 0.1 on a saturation binding curve and/or an agonist dose–response curve.

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Characteristics of Allosterism in Drug Action

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(A)

Allosteric (α = 0.1)

Fract. Max. Resp.

1.2 1.0 0.8 0.6 0.4 0.2 0.0 –3

–2

–1

0

1

2

3

4

3

4

(B)

Simple Competitive Fract. Max. Resp.

1.2 1.0 0.8 0.6 0.4 0.2 0.0

–3

–2

–1

0 1 Log ([A]/KA)

2

Figure 3 Effects of (A) an allosteric antagonist and (B) simple competitive (orthosteric) antagonist on dose–response curves to a receptor probe (either agonist or radioligand). (A) For the allosteric antagonist, a ¼ 0.1; a maximal 10-fold shift to right is observed. (B) For the simple competitive antagonist, theoretically inﬁnite shifts to the right can be obtained.

It can be seen that the maximal dextral displacement of the curve is 10-fold in accordance with the allosteric nature of the effect. Therefore, unlike a competitive antagonist where the dextral displacement continues in a uniform manner according to the Schild equation (dose-ratio ¼ ([B]/KB) þ1, where [B] is the concentration of antagonist), the dose-ratios for an allosteric antagonist are predicted by the equation (41): Dose-ratio ¼

½Bð1 aÞ þ1 a½B þ KB

ð1Þ

It can be seen from Eq. (1) that, unlike the competitive situation where the dose-ratio ! 1 as [B] ! 1, as the concentration of antagonist approaches very high values ([B] ! 1), the dose-ratio approaches the limit 1/a (the dextral displacement of the dose–response curves reaches a

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Kenakin

maximal value). For simple competitive antagonism, usually a Schild regression according to the equation: LogðDR 1Þ ¼ Log½B LogKB

ð2Þ

is utilized to quantify antagonism. Under these circumstances, a linear regression of unit slope is used to calculate the intercept value which itself is the equilibrium dissociation constant of the antagonist–receptor complex (KB). If the antagonism is saturable, then at some point, as the concentration of antagonist added to the assay is increased, the effect will diminish and deviate from that predicted by the Schild regression Eq. (2). In fact, the Schild regression for an allosteric antagonist will be predicted by: ½Bð1 aÞ LogðDR 1Þ ¼ Log ð3Þ a½B þ KB This results in a curvilinear Schild regression the shape of which is dependent upon the magnitude of a (Fig. 4). Therefore, the observation of a saturable antagonism is indicative of allosteric and not simple competitive receptor blockade. While theory predicts a curved Schild regression, in practical terms it may not be possible to deﬁne deviation from linearity and an apparently linear Schild regression with a slope <1 may be observed experimentally (Fig. 4). It is important to note that, over normally utilized concentrations of antagonist (between [B]/KB ¼ 2 and 100), the slope of the Schild regression will be inversely proportional to the magnitude of a. This

Figure 4 Schild regressions for allosteric antagonists with differing cooperativity values (a ¼ 0.3–0.001). Curvilinear regressions are predicted (dotted lines) which may appear to be linear with a slope less than unity if there is a paucity of datapoints. The magnitude of the slope is inversely proportional to the value of a.

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provides a useful practical test for allosterism, namely the dependence of the antagonism on the nature of the probe. Speciﬁcally, different agonists may have different values of a for the allosteric antagonist and therefore, the slope of the experimental Schild regression may vary with the agonist used to make the measurement. This behavior is not consistent with simple competitive antagonism where the nature of the agonist used for the measurement is immaterial, i.e., the slope of regression should be unity for all antagonists. It should also be noted that for allosteric antagonists of low a values (<0.01), antagonism will appear to be simple competitive over a large concentration range of the antagonist ([B]/KB 100) and ostensibly be consistent with simple competitive antagonism. However, since the magnitude of a is probe dependent, other agonists should be tested to unveil heterogeneity in the antagonistic effects resulting from allosterism. Figure 3 shows the rigorous experiment, namely the testing of a full range of concentrations of probe against a full range of concentrations of antagonist (effects of antagonist on full dose–response curves). Often this is not feasible and an alternative format, namely the testing of a range of concentrations of antagonist on the effects of a single concentration of probe (agonist or radioligand), is used. This latter format, referred to as a displacement experiment, is common for binding studies as full radioligand saturation curves are difﬁcult and expensive to obtain. The relationship between displacement and full dose–response curves is shown in Figure 5 where it can be seen that the saturability of the allosteric effect (for Fig. 5A, a ¼ 0.1) leads to an inability of the antagonist to completely reverse the effects of the probe. This is in contrast to the effect of a simple competitive antagonist that will always reduce the probe stimulus to baseline values (Fig. 5B). Therefore, a characteristic of allosteric mechanism in displacement curves is a failure to attain baseline values. However, there are conditions where baseline levels will be achieved, as in the case of low concentrations of probe and low values of a. Therefore, this is a one-way observation in that, if baseline is not achieved, then allosterism in implied; if it is not, allosterism still may or may not be operative. The maximal degree of antagonism is inversely proportional to the magnitude of a and, as such, is dependent on the strength of probe stimulus. Figure 6 shows different maximal displacement effects of an allosteric ligand of a ¼ 0.1 when utilized against different strengths of probe signal (in this case concentration of either agonist or radioligand). It can be seen that, the more intense the probe signal, the more incomplete is the displacement by the allosteric antagonist. Another kind of dissociation of information can occur with allosteric ligands when they are studied with different assays, speciﬁcally binding and functional assays. For orthosteric ligands, the presence of the ligand in the binding pocket essentially precludes binding of the agonist (or radioligand) and function generally mirrors binding. However, allosteric binding

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(A)

Allosteric

Fract Max. Resp

1.0

Displacement

0.8 0.6 0.4 0.2 0.0 –3

–2

–1 0 1 Log ([A]/KA)

2

–2

4

Simple Competitive

(B) 1.0 Fract Max. Resp

0 2 Log ([B]/KB)

Displacement

0.8 0.6 0.4 0.2 0.0 –3

–2

–1 0 1 Log ([A]/KA)

2

–2

0 2 Log ([B]/KB)

4

Figure 5 Relationships between the shifts produced by (A) allosteric and (B) competitive antagonists on full dose–response curves and corresponding displacement curves produced by testing one level of receptor stimulation (or initial level of radioligand binding) against a range of concentrations of antagonist (displacement curves as shown in the right-hand panels). Note how displacement curves may not indicate complete displacement to zero ordinate values for allosteric antagonists.

mechanisms are permissive in that the binding of the allosteric ligand does not necessarily modify the occupancy of the receptor by the probe but may modify the physiological result of the binding by the probe. A model of allosteric function described by Hall (66) can be used to illustrate cases where a ligand-mediated inhibition of receptor function is not necessarily concomitant with a decrease in radioligand binding (see Chapter 3). It is the fact that binding and function measure the prevalence of different receptor species that leads to possible dissimulation between binding and function. As discussed previously, such effects have been reported as in the functional noncompetitive blockade of glutamate

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1.0 % Max. Resp.

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α-dependent [A]/KA dependent 10

0.8 0.6 1 0.4

0.1 [A]/KA=0.01

0.2 0.0 –3

–1

1 Log ([B]/KB)

3

5

Figure 6 Effects of various parameters on displacement curves to allosteric antagonists. The potency of the antagonist, as indicated by the abscissal location parameter of the displacement curve, is inversely proportional to the strength of the initial stimulus given the receptor (either initial agonist response or initial binding of radioligand tracer). The maximal displacement is a function of the magnitude of the cooperativity constant a and the strength of initial receptor stimulation.

receptors by clopropan[b]chromen-1a-carboxylic acid ester with no comcomitant effects on glutamate (43). THE FUTURE OF ALLOSTERIC LIGANDS AS DRUGS In the past two decades, binding assays have primarily been used to discover new drug entities. Now that the technology has advanced to the point where the ability to screen in functional receptor systems has equaled and/or surpassed binding in terms of throughput and efﬁciency, an increase in the number of functionally allosteric drugs will be seen (67). Therefore, it is imperative that pharmacologists be able to quantify the activity of these new drug entities and appreciate their special properties as they are applied therapeutically.

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3. Dragic T, Litwin V, Allaway GP, et al. HIV-1 entry into CD4þ cells is mediated by the chemokine receptor CC CCR-5. Nature 1996; 381:667–673. 4. Alkhatib G, Combadiere C, Broder CC, et al. RANTES, MIP-la, MIP-lb receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 1996; 272:1955–1958. 5. Choe H, Farzan M, Sun Y, et al. 1996. The b-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 1996; 85:1135–1148. 6. Doranz BJ, Rucker J, Smyth Y, et al. A dual-tropic primary HIV-1 isolate that uses fusin and the b-chemokine receptors CKR-5, CKR-3 and CKR-2b as fusion cofactors. Cell 1996; 85:1149–1158. 7. Rucker J, Edinger AL, Sharron M, et al. Utilization of chemokine receptors, orphan receptors, and herpesvirus encoded receptors by diverse human and simian immunodeﬁciency viruses. J Virol 1997; 71:8999–9007. 8. Doms RW, Peiper SC. Beyond receptor expression: the inﬂuence of receptor conformation, density, and afﬁnity in HIV-1 infection. Virology 1997; 235: 179–190. 9. Lee B, Sharron M, Blanpain C, et al. Epitope mapping of CCR5 reveals multiple conformational states and distinct but overlapping structures involved in chemokine and coreceptor function. J Biol Chem 1999; 274:9617–9626. 10. Picard L, Simmons G, Power CA, Meyer A, Wiess RA, Clapham PR. Multiple extracellular domains of CCR-5 contribute to human immunodeﬁciency virus type 1 entry and fusion. J Virol 1997; 71:5003–5011. 11. Atchison RE, Gosling J, Monteclaro FS, et al. Multiple extracellular elements of CCR5 and HIV-1 entry: dissociation from response to chemokines. Science 1996; 274:1924–1926. 12. Bieniassz PD, Fridell RA, Aramori I, Ferguson SS, Caron MG, Cullon BR. HIV-1 induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor. EMBO J 1997; 16:2599–2609. 13. Ross T, Bieniasz PD, Cullen BR. Role of chemokine receptors in HIV-1 infection and pathogenesis. Adv Virus Res 1999; 52:233–267. 14. Kwong PD, Wyatt R, Robinson J, Sweet RW, Sodroski J, Hendricks WA. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 1998; 393:648–659. 15. Rizzuto CD, Wyatt R, Hernandez-Ramos N, et al. A conserved HIV gp120 glycoprotein structure involved in chemokine receptor binding. Science 1988; 280:1949–1953. 16. Smyth RJ, Yi Y, Singh A, Collman RG. Determinants of entry cofactor utilization and tropism in a dualtropic human immunodeﬁciency virus type 1 isolate. J Virol 1998; 72:4478–4484. 17. Doranz BJ, Lu Z-H, Rucker J, et al. Two distinct CCR5 domains can mediate coreceptor usage by human immunodefﬁciency virus type 1. J Virol 1997; 71:6305–6314. 18. Baba M, Nishimura O, Kanzaki N, et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti- HIV-1 activity. Proc Natl Acad Sci USA 1999; 96:5698–5703. 19. Finke G, Oates B, Mills SG, et al. Antagonists of the human CCR5 receptor as anti-HIV-1 agents. Part 4: synthesis and structure–activity relationships for

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36. Kenakin TP. Efﬁcacy at G protein coupled receptors. Nat Revs Drug Discov 2002; 1:103–109. 37. Kenakin T, Onaran O. The ligand paradox between afﬁnity and efﬁcacy: can you be there and not make a difference? Trends Pharmacol Sci 2002; 23:275–280. 38. Burgen ASV. Conformational changes and drug action. Fed Proc 1966; 40:2723–2728. 39. Ehlert FJ. Estimation of the afﬁnities of allosteric ligands using radioligand binding and pharmacological null methods. Mol Pharmacol 1988; 33:187–194. 40. Jakubic J, Bacakova L, Lisa V, El-Fakahany EE, Tucek S. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors. Mol Pharmacol 1997; 52:172–179. 41. Christopolous A, Kenakin TP. G-protein coupled receptor allosterism complexing. Pharmacol Rev 2002; 54:323–374. 42. Hejnova L, Tucek S, El-Fakahany EE. Positive and negative allosteric interactions on muscarinic receptors. Eur J Pharmacol 1995; 291:427–430. 43. Litschig S, Gasparini F, Rueegg D, et al. CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding. Mol Pharmacol 1999; 55:453–461. 44. Jakubic J, Bacakova L, Lisa V, El-Fakahany EE, Tucek S. Activation of muscarinic acetylcholine receptors via their allosteric binding sites. Proc Natl Acad Sci USA 1996; 93:8705–8709. 45. Moore JP, Trkola A, Dragic T. Co-receptors for HIV-1 entry. Curr Opin Immunol 1997; 9:551–562. 46. Nagasaw T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996; 382:635–638. 47. Tachibana K, Hirota S, Iizasa H, et al. The chemokine receptor CXCR4 is essential for vascularization of the gastrointestinal tract. Nature 1998; 393: 591–594. 48. Zou YR, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 1998; 393:595–599. 49. Sachpatzidis A, Benton BK, Manfredis JP, et al. Identiﬁcation of allosteric peptide agonists of CXCR4. J Biol Chem 2003; 278:896–907. 50. Heveker N, Montes M, Germeroth L, et al. Dissociation of the signalling and antiviral properties of SDF-1-derived small peptides. Curr Biol 1988; 8: 369–376. 51. Ghanouni P, Grycynski Z, Steenhuis JJ, et al. Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta (2) adrenergic receptor. J Biol Chem 2001; 276: 24,433–24,436. 52. Bursavich MG, Rich DH. Designing non-peptidomimetics in the 21st century: inhibitors targeting conformational ensembles. J Med Chem 2001; 45:541–558. 53. Stephenson RP. A modiﬁcation of receptor theory. Br J Pharmacol 1956; 11:379–393. 54. Kenakin TP. Ligand-selective receptor conformations revisited: the promise and the problem. Trends Pharmacol Sci 2003; 24:346–354.

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55. Roettger BF, Ghanekar D, Rao R, et al. Antagonist-stimulated internalization of the G protein-coupled cholecystokinin receptor. Mol Pharmacol 1997; 51: 357–362. 56. Blanpain C Vanderwinden J-M, Cihak J, et al. Multiple active states and oligomerization of CCR5 revealed by functional properties of monoclonal antibodies. Mol Biol Cell 2002; 13:723–737. 57. Tucek S, Proska J. Allosteric modulation of muscarinic acetylcholine receptors. Trends Pharmacol Sci 1995; 16:205–212. 58. Fawzi AB, MacDonald D, Benbow LL, et al. SCH-202676: an allosteric modulator of both agonist and antagonist binding to G protein-coupled receptors. Mol Pharmacol 2001; 59:30–37. 59. Bartus RT, Dean RL, Beer B, Lippa AS. The cholinergic hypothesis of geriatric memory dysfunction. Science 1982; 217:408–417. 60. Flicker L. Acetylcholinesterase inhibitors for Alzheimer’s disease. Br Med J 1999; 318:615–616. 61. Nordberg A, Svensson AL. Cholinesterase inhibitors in the treatment of Alzheimer’s disease: a comparison of tolerability and pharmacology. Drug Saf 1998; 19:465–480. 62. Maelicke A, Schrattenholz A, Samochocki M, Radina M, Albuquerque EX. Allosterically potentiating ligands of nicotinic receptors as a treatment strategy for Alzheimer’s disease. Behav Brain Res 2000; 113:199–206. 63. Rogers SL, Farlow MR, Doody RS, Mohs R, Friedhoff LT. Donepezil Study Group A 24-week, double-blind, placebo-controlled trial of donepezil in patients with Alzheimer’s disease. Neurology 1998; 50:136–145. 64. Maelicke A, Albuquerque EX. New approach to drug therapy of Alzheimer’s dementia. Drug Discov Today 1996; 1:53–59. 65. Krause RM, Buisson B, Bertrand S, et al. Ivermectin: a positive allosteric effector of the a7 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 1998; 53:283–294. 66. Hall DA. Modeling the functional effects of allosteric modulators at pharmacological receptors: an extension of the two-state model of receptor activation. Mol Pharmacol 2000; 58:1412–1423. 67. Rees S, Morrow D, Kenakin T. GPCR drug discovery through the exploitation of allosteric drug binding sites. Receptors Channels 2002; 8:261–268.

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3 Predicting Dose–Response Curve Behavior Mathematical Models of Allosteric Receptor–Ligand Interactions David A. Hall Respiratory Pharmacology, Respiratory and Inflammation Center of Excellence for Drug Discovery, GlaxoSmithKline, Stevenage, Herts, U.K.

INTRODUCTION Mathematical modeling is simply the process of formulating real-world problems or processes into mathematical equations whose behavior mimics the key features of those systems. The model may then be used qualitatively to gain further insight into the behavior of the system or, more quantitatively, for hypothesis testing. In either case, a comparison of the behavior of the model with that of the experimental system tests the appropriateness of the model. The behaviors of the model may also suggest further experiments to explore the system under study (and to further test the correspondence between the model and reality). Some of these may not have been previously considered. Indeed, it can be argued that without a mathematical formulation, it is not possible to test rigorously whether a scientiﬁc theory is consistent with experimental observations. The modeling process is, of course, iterative with observations driving reﬁnement of the mathematical model and new behaviors of the model potentially suggesting further experimental avenues. A corollary of the previous paragraph is that many methods of data analysis are, in fact, the quantitative comparison of the predictions of a 39

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mathematical model with experimental data. A simple and familiar example of this is the analysis of saturation equilibrium binding data. The reversible binding of a ligand (L) to a single site on a receptor (R) is described by the chemical equation L þ R $ LR. From the deﬁnition of the equilibrium constant and the requirement that the total mass of the receptor is conserved, it is possible to derive a relationship between the concentrations of L and LR present in the system at equilibrium: [LR] ¼ [R]T[L]/(KD þ [L])—the Langmuir binding isotherm. Thus, modeling the behavior of this system at equilibrium predicts that the binding of a ligand to a single population of noninteracting binding sites is a rectangular hyperbolic function of the ligand concentration (nonspeciﬁc binding is not a feature of the Langmuir model). Fitting the more general, but less physically meaningful, Hill equation, [LR] ¼ [R]T[L]n/(Kn þ [L]n), to the experimental data allows the appropriateness of the Langmuir model to be tested (by showing that the Hill coefﬁcient does not differ from unity). Once the experimental data have been shown to be consistent with the Langmuir model, the midpoint of the ﬁtted hyperbola can be assumed to be an estimate of the afﬁnity of the ligand for that site. If the Hill coefﬁcient is not unity then it becomes necessary to determine how the system differs from the model. This may be as simple as inappropriate experimental conditions preventing equilibrium from being reached before the reaction is terminated, or the system may, in fact, contain more than one receptor or a receptor that has several interacting binding sites for the ligand. The fundamental characteristic of allosteric interactions, which forms the basis of all mathematical models of allosterism, is that allosteric ligands bind to a site on the receptor that is spacially distinct from that of the natural ligand (which is often termed the orthosteric site). In contrast to a competitive ligand, the effects of which are purely steric, an allosteric ligand actually changes the afﬁnity of the receptor for the orthosteric ligand by inducing a conformational change in the receptor protein. This change in afﬁnity can, of course, be in either direction. The change in afﬁnity is accompanied by possibly the most diagnostic effect of an allosteric ligand on the binding of an orthosteric ligand—a change in its binding kinetic constants (usually measured as a change in the dissociation rate constant) (1). Competitive ligands cannot inﬂuence each other’s dissociation kinetics as they are never bound simultaneously to the receptor. Given the above, an allosteric protein must contain multiple ligandbinding sites. These sites may bind the same ligand (with identical or different afﬁnities) or may bind different ligands. The interaction in the former case is termed homotropic cooperativity, while the latter is termed heterotropic cooperativity. As the subject of this book is allosterism in drug discovery, I will concentrate primarily on the mathematical models that describe heterotropic interactions; however, little modiﬁcation is required to apply any of these models to homotropic interactions. Once we deﬁne allosteric interactions as occurring between spacially distinct sites, a further

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property of allosteric interactions follows. The effects of allosteric modulators should be saturable since it is possible for allosteric ligands to saturate their respective binding sites on the receptor simultaneously. The basis of a mathematical model to describe a pharmacological system will almost invariably be a system of linked elementary (that is bimolecular) binding reactions. Models including functional responses may also include receptor isomerization steps, if based on the two-state model; a transducer function, if formulated using the operational model; or binding of the receptor to a transducer protein (e.g., the ternary complex model for G-protein–coupled receptors) (2–7). In the majority of cases, it is the equilibrium behavior of the system that is of interest. An equilibrium constant can then be deﬁned for each of the binding reactions that make up the system. In a system involving allosteric interactions, it is possible to deﬁne two classes of equilibrium binding constants: the unconditional equilibrium constants, which characterize the formation of each binary ligand–receptor complex, and the conditional equilibrium constants, which characterize the binding of the ligand to a further site on a preexisting complex. The ratio of the conditional and unconditional binding constants for a given ligand (termed an allosteric constant) is a measure of the free energy coupling (i.e., cooperativity) between the binding of the two ligands. Because there can be no net free energy change in a system at equilibrium, this free energy coupling must be the same in both directions: the effect of A on the afﬁnity of B is the same as the effect B on the afﬁnity of A. This requirement was termed the principle of conservation of free energy by Weber (8). However, as Weber pointed out, it is a simple consequence of the thermodynamic constraints that apply to a system at equilibrium. If the change in afﬁnity were not reciprocal, there would be a net free energy change on traversing the reaction cycle and it would not then represent an equilibrium. The relationship between the concentration of the ligands and their complexes can now be derived by applying the principle of conservation of mass to the receptor and assuming that the reactions take place under pseudo-ﬁrst order conditions. The latter is the assumption that the ligands are present in sufﬁcient excess over the receptor that the formation of the complexes leaves their free concentrations unchanged. This assumption is generally valid for small molecules (unless they have very high afﬁnity for the receptor), but may not be valid for transducer or effector proteins, such as G-proteins. I will explore systems in which this is not the case in the section on modeling the functional effects of allosteric modulators. In models in which each ligand (Li) binds speciﬁcally to only a single binding site on the receptor, the relationship between the concentration of Li and its complexes is a rectangular hyperbola. That is, it is of the form y¼

a þ b½Li c þ d½Li

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where y is the fractional occupancy of Li or a sum of the concentrations of receptor complexes containing Li, and a, b, c, and d are the functions of the equilibrium constants and the concentrations of the other ligands in the system. When plotted against log[Li], Eq. (1) has two horizontal asymptotes: a/c when [Li] ¼ 0 and b/d when [Li] ! 1. The midpoint of Eq. (1) is c/d and its Hill coefﬁcient is 1. These expressions are derived in part 1 of the Appendix [see also Pardo et al. (9)]. Thus, these parameters can be derived conveniently from equations of the form of Eq. (1) by inspection. For systems with n sites for a given ligand, the highest power of the ligand concentration in the binding isotherm is [L]n. This highlights an interesting contrast between homotropic and heterotropic interactions. Cooperativity between identical ligand molecules results in binding curves with Hill coefﬁcients that differ from unity (apart from certain special cases). That is, they can exhibit cooperative binding. However, the interaction between n different ligands at n speciﬁc binding sites results in theoretical binding curves whose Hill coefﬁcients are unity (which will be illustrated in the two-site case below). In the rest of this chapter, I shall start by reviewing the mathematical models (reaction schemes) of allosteric modulation that have been presented previously. I will initially describe those models that deal only with binding reactions. I will then consider those that have attempted to model the effects of allosteric interactions on functional responses. Finally, I will consider a relatively unexplored aspect of allosteric modulation in functional systems—the inﬂuence of receptor reserve. By necessity, my exploration of the behavior of any particular model will be brief. I would encourage readers to transcribe the equations into their favorite spreadsheet package—the behaviors of a model can only fully be appreciated by the systematic exploration of the effects of its parameters on simulated binding isotherms or concentration–effect curves. One area I shall not consider is modeling the effects of allosteric compounds on binding kinetics. This is primarily because a general treatment of even a simple model has no analytical solution and requires recourse to numerical methods. For a treatment of some of the cases where an analytical solution is possible see Lazareno and Birdsall (10). The reader should also note that systems that do not reach equilibrium can behave quite differently from the predictions of the equilibrium models outlined below (11,12). Finally, a brief note on the conventions I will use in this chapter. For consistency, I will use association constants to characterize binding reactions throughout (apart from the operational model derivation where dissociation constants are more consistent with formalism). This is primarily from personal preference, but also because the resulting formulae are somewhat ‘‘neater’’ than those derived using dissociation constants. Also, I will denote the radioligand in a binding model or primary orthosteric agonist in a functional model as ligand A and the ﬁrst allosteric ligand as ligand B. Further ligands will simply follow in alphabetical order; their binding site(s) should be clear

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from the associated reaction scheme. This convention is, of course, entirely arbitrary and merely for convenience rather than a requirement of the mathematics of the models.

MODELING ALLOSTERIC EFFECTS ON LIGAND BINDING The simplest and the most frequently used model of an allosteric interaction between two ligands is the ternary complex model (Fig. 1A). This model was ﬁrst proposed by Stockton et al. (13) to explain the effects of gallamine on ligand binding to muscarinic receptors. Its behavior was discussed in some

Figure 1 The potential reaction schemes in a system consisting of two ligands (A and B) and a receptor with two binding sites. For clarity unbound ligands are not shown. (A) Each ligand is speciﬁc for one of the two sites. K and M are the unconditional association constants for A and B, respectively, and a is the cooperativity constant. (B) Ligand A is speciﬁc for the orthosteric site; B can bind to both sites. K is the unconditional association constant for A. M and N are the unconditional association constants of B for the allosteric and orthosteric sites, respectively. a is the allosteric constant governing the interaction between A and B. b is the allosteric constant governing the interaction of B with itself. (C) Ligand A binds to both sites, ligand B is speciﬁc. K and L are the unconditional association constants of A and M is the association constant of B. a is the allosteric constant governing the interaction between A and B. c is the allosteric constant governing the interaction of A with itself. (D) Both ligands bind to both sites. K and L are the unconditional association constants of A, and M and N are the unconditional association constants of B. a and d are the allosteric constants governing the interaction between A and B. b and c are the allosteric constants governing the interaction of A and B, respectively, with themselves.

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detail by Ehlert (14); however, I shall also outline its key properties below. Since this is the most basic model of an allosteric interaction, it underpins our expectations of allosteric behavior. In this model, the receptor has two binding sites and the two ligands are each speciﬁc for one of these sites. Interactions between ligands that are consistent with this model have been reported at a number of receptors (see the other chapters in this book, Refs. 15–17 and references therein). The theoretical equilibrium-binding isotherm for this model is ½AR þ ½ARB K½Að1 þ aM½BÞ ¼ ½RT 1 þ M½B þ K½Að1 þ aM½BÞ

ð2Þ

where, K ¼ ½AR=ð½A½RÞ and M ¼ ½RB=ð½B½RÞ are the unconditional association constants of A and B, respectively, and a ¼ ½R½ARB=ð½AR½RBÞ is the allosteric constant that quantiﬁes the cooperativity of the interaction between A and B. Equation (2) is of the same form as Eq. (1) and thus, by inspection, the apparent afﬁnity (KD,app) of A is KD;app ¼

1 þ M½B Kð1 þ aM½BÞ

which is itself a hyperbola, in this case in [B], and takes the value KD;app ¼ 1=K, when [B] ¼ 0 and KD;app ¼ 1=aK when [B] ! 1 (i.e., when B saturates its binding site). Therefore, a deﬁnes the maximal change in the binding afﬁnity of A in the presence of B (or vice versa). When a > 1, the afﬁnity of A at saturating [B] is greater than in the absence of B—the cooperativity is positive. When a < 1, the afﬁnity of A at saturating [B] is less than in the absence of B giving negative cooperativity. When a ¼ 1, B has no effect on the equilibrium binding of A. This phenomenon, termed neutral cooperativity, has been observed at muscarinic cholinergic receptors and at c-aminobutyric acid (GABAA) receptors—the benzodiazepine receptor neutral antagonists (10,18–20). Interestingly, while neutrally cooperative ligands do not affect the equilibrium binding of A, they can still affect its binding kinetics [e.g., Lazareno and Birdsall (10)]. In this case, the changes in the magnitude of the association and dissociation rate constants are identical and cancel out in the equilibrium constant, which is the ratio of the two. Of course, binding experiments are most commonly performed by titrating B against a constant concentration of A rather than by performing multiple saturation curves to A. To investigate the model’s predictions in this situation, it is helpful to rearrange Eq. (2) as follows: ½AR þ ½ARB K½A þ aKM½A½B ¼ ½RT 1 þ K½A þ M½Bð1 þ aK½AÞ

© 2006 by Taylor & Francis Group, LLC

ð2aÞ

Predicting Dose–Response Curve Behavior

45

In this form, it is clear [again by analogy to Eq. (1)] that the midpoint of the B titration curve, [B]50, is ½B50 ¼

1 þ K½A Mð1 þ aK½AÞ

(note the close analogy between this and the expression for KD,app above) and the fractional occupancy of A at saturating [B] is aK½A=ð1 þ aK½AÞ. Depending on the value of a, this can be greater than (a > 1), the same as (a ¼ 1), or less than (a < 1) the control fractional occupancy of A, K½A=ð1 þ K½AÞ (Fig. 2A). The maximal level of inhibition only approximates 100%, and therefore the behavior of a competitive ligand when a 1, that is, in cases of very strong negative cooperativity. The maximal level of inhibition is, of course, also dependent on [A], suggesting that if [A] is sufﬁciently high, the noncompetitive behavior of B will be revealed (Fig. 2B). However, this is subject to practical constraints since the maximal value of [B]50 is 1/aM. This may require the use of concentrations of B that are not practically attainable to saturate the receptor. A notable variant of this model is that postulated for the interaction between alcuronium and muscarinic ligands (11). In this case, the allosteric ligand is thought to bind at a site that occludes the ligand-binding pocket. This prevents binding of ligands to unoccupied orthosteric sites, but also prevents dissociation of the orthosteric ligand when it is already bound. This mechanism corresponds to Figure 1A with the A þ RB $ ARB reaction deleted. It is therefore a linear rather than cyclic reaction scheme. The equilibrium behavior of this system is identical with that of Figure 1A; it is the pre–steady-state behavior that differs. Importantly, high concentrations of the allosteric modulator can very markedly delay the approach to equilibrium in this scheme (and for some cases of Fig. 1A itself), potentially confounding attempts to measure binding at steady state. As shown in Figure 1B–D, there are three other possible reaction schemes that can describe the interaction between two ligands at a receptor with two binding sites. These allow one or both of the ligands to bind to both binding sites on the receptor and hence to interact competitively as well as allosterically. Actually, Figure 1B and C are mathematically identical since the deﬁnition of the allosteric and orthosteric ligand is arbitrary (and therefore interchangeable) rather than a speciﬁc feature of the model. I have shown the two cases separately to distinguish between the physical situation in which the secondary ligand (B) can also bind to the orthosteric site (Fig. 1B) and that in which the primary ligand (A) binds to two sites and the secondary ligand is speciﬁc for one of these (Fig. 1C). In either case, the binding sites cannot be identical. The mechanism in Figure 1B has been proposed, for example, for the interaction of amiloride analogs and spiperone at D2-like and D1 dopamine receptors (21,22). In fact, the schemes in

© 2006 by Taylor & Francis Group, LLC

46

Hall

Figure 2 (A) The effect of a on the titration curve to B generated by the reaction scheme in Figure 1A. The concentration of A is 1/K (i.e., the radioligand is present at KD). The afﬁnity constants are K ¼ M ¼ 1. (B) The effect of [A] on the titration curve to B generated by the reaction scheme in Figure 1A. The afﬁnity constants are K ¼ M ¼ 1 and a ¼ 0.01. Note as [A] is increased the maximal level of inhibition by B decreases while the midpoint of the titration curve shifts rightward (to a maximum value of 1/aM).

Figure 1A–C are special cases of Figure 1D (15). As the interactions become more complex, it becomes increasingly difﬁcult to deﬁne an allosteric site unambiguously (hence the use of the terms primary and secondary ligands). In Figure 1D, both binding sites are orthosteric. The equations describing

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47

the binding isotherms predicted by these schemes are shown in Table 1. Midpoints are given only where they can be meaningfully deﬁned for the system (i.e., for those that do not have the potential to generate bell-shaped curves). In Figure 3, I have illustrated some of the possible behaviors of schemes 1B and 1D (the situation described by scheme 1C is likely to be quite rare). The most striking feature of the equation for the binding isotherm derived from scheme 1B is that the denominator is no longer linear in [B]. This means that titration curves for B can be obtained that are steeper or ﬂatter than the standard mass–action relationship. Indeed, the appropriate choice of allosteric constants can result in biphasic and even bell-shaped curves. The bell-shaped curves are an a-driven phenomenon. They result when the cooperativity between ligands is positive and the afﬁnity of B for the allosteric site is greater than that for the orthosteric site. The mathematical condition for bell-shaped curves is a > 1 þ N/M, in which case the peak of the curve occurs at

½B ¼

bN þ

pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ bNðNðb aÞ þ aMða 1ÞÞ abMN

These expressions are derived in part 2 of the Appendix. Biphasic titration curves are largely a b-driven phenomenon and occur when the cooperativity of B binding is negative (they can also occur when one of M or N is much greater than the other). The possible behaviors of reaction scheme 1D are similar to those of 1B with the added complexity that the binding curves of A can also deviate from those of a simple mass–action isotherm. As with Figure 1B, the titration curves can be made to ﬂatten simply by judicious choice of the unconditional association constants as well as by modifying the allosteric constants. This is an example of the well-known result that Hill coefﬁcients less than unity can be caused either by negative cooperativity or by the presence of two independent sites with different afﬁnities. Bell-shaped titration curves are again possible, but under more restricted circumstances. The criterion for bell-shaped curves is cKLðaKM þ dLNÞ½A2 þ 2cKLðM þ NÞ½A þ ðK þ LÞðM þ NÞ ðaKM þ dLNÞ < 0 (see part 3 of the Appendix for the derivation). Apart from its complexity, the most striking feature of this expression is its dependence on [A]. The precise mathematical condition for bell-shaped curves is that [A] is less than the solution to the quadratic equation formed when the above expression equals zero. However, this is rather complicated. A simpler, approximate condition pﬃﬃﬃﬃﬃﬃﬃﬃﬃ is that bell-shaped titration curves cannot occur when ½A 1= cKL

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48

Table 1 Binding Isotherms of the Reaction Schemes Presented in Figure 1 Fractional occupancy of Aa

KD,app

[B]50b

K½Að1 þ aM½BÞ 1 þ M½B þ K½Að1 þ aM½BÞ

1 þ M½B Kð1 þ aM½BÞ

1 þ K½A Mð1 þ aK½AÞ

K½Að1 þ aM½BÞ

1 þ ðM þ NÞ½B þ bMN½B2 Kð1 þ aM½BÞ

2

1 þ ðM þ NÞ½B þ bMN½B þ K½Að1 þ aM½BÞ ðK þ LÞ½A þ aKM½A½B þ 2cKL½A2 2ð1 þ ðK þ LÞ½A þ cKL½A2 þ M½Bð1 þ aK½AÞÞ ðK þ LÞ½A þ 2cKL½A2 þ ðaKM þ dLNÞ½A½B 2ð1 þ ðK þ LÞ½A þ cKL½A2 þ ðM þ NÞ½B þ bMN½B2 þ ðaKM þ dLNÞ½A½BÞ

sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ M½B cKL

1 þ ðK þ LÞ½A þ bKL½A2 Mð1 þ aK½AÞ

sﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 þ ðM þ NÞ½B þ bMN½B2 cKL

The factor of 2 in the demoninator of 1C and 1D occurs because the concentration of binding sites is twice the total receptor concentration in a two-site system. Where no formula is stated the curves generated can be bell-shaped. In this case the ‘‘midpoint’’ of the curve is difﬁcult to deﬁne and has little descriptive value.

Hall

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Predicting Dose–Response Curve Behavior

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Figure 3 (A) The effect of a in the reaction scheme in Figure 1B on titration curves to B. When a 1 þ N/M the effect of B is only inhibitory. Under these conditions the titration curves may be steep, ﬂat, biphasic (not shown) or have Hill coefﬁcients of unity. The observed behaviors depend largely on the ratio N/M. When a > 1 þ N/M bell-shaped titration curves result. When a1 þ N=M an extended plateau region occurs around pﬃﬃﬃﬃﬃﬃﬃﬃﬃthe peak of the curve. Other parameters were K ¼ M ¼ N ¼ 1, b ¼ 1, ½A ¼ 1= cKL. (B) The effect of b in the reaction scheme in Figure 1B on titration curves to B. The effects of b are somewhat subordinate to those of a. Thus, when a > 1 þ N/M, bell-shaped curves still result at all values of b, although the width and height of the peak are modiﬁed (if b is large the peak is lower and narrower) (not shown). When a 1 þ N/M, b causes the curves to steepen pﬃﬃﬃﬃﬃﬃﬃﬃﬃ or become biphasic. Other parameters were K ¼ M ¼ N ¼ 1, a ¼ 1, ½A ¼ 1= cKL. (C) Variation in B titration curve shape with [A] in the reaction scheme in Figure 1D. Note the transition to a biphasic curve when fractional occupancy p isﬃﬃﬃﬃﬃﬃﬃﬃﬃ 0.5. Other parameters were K ¼ L ¼ M ¼ N ¼ 1, a ¼ 30, b ¼ c ¼ d ¼ 1, Kapp ¼ 1= cKL.

(the midpoint of the control curve), i.e., when the control fractional occupancy 0.5 (see Fig. 3 and part 3 of the Appendix). Above this threshold, the same values of a result in biphasic titration curves. This result is intuitively reasonable, as bell-shaped curves require a positively

© 2006 by Taylor & Francis Group, LLC

50

Hall

cooperative interaction between the two ligands. When the radioligand occupies a signiﬁcant fraction of both sites, this allosteric interaction will be compromised. The last models of the two binding site systems that I will deal with are the cases in which there are three ligands present. These correspond to two experimental approaches. In the ﬁrst (Fig. 4A), the interaction between an allosteric ligand and an unlabeled orthosteric ligand are to be determined (e.g., Refs. 10,13,23). In the second, the interaction between the two allosteric ligands is probed to determine whether they interact at a common binding site (Fig. 4B) [e.g., Leppik et al. (24)]. The latter will be compared with the predictions of a three-site, three-speciﬁc ligand model (Fig. 4C) (18). In each case, the third ligand in the system is the radioligand. Expressions for the theoretical binding isotherms for these three models are given in Table 2. These models all result in binding isotherms of the form of Eq. (1), so the limits and midpoints of the curves can be derived by inspection.

Figure 4 Selected three ligand systems. (A) The interaction of an allosteric ligand (B) with two ligands (A and C) that compete at the orthosteric site. K, M, and N are the unconditional association constants of A, B, and C, respectively. a and b are the allosteric constants describing the interaction of B with A and C, respectively. (B) The interaction between two allosteric ligands (B and C) that bind to the same binding site and an orthosteric ligand (A). K, L, and M are the unconditional association constants of A, C, and B, respectively, and a and c are the allosteric constants describing the interaction of B and C, respectively, with A. (C) The interaction of three ligands through three different binding sites. K, M, and N are the unconditional association constants of A, B, and C, respectively. a, b, and c are the allosteric constants for the pairwise interactions between A and B, A and C, and B and C, respectively. d is the allosteric constant that characterizes the change in free energy coupling between the binding sites when all three ligands are bound.

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Fractional occupancy of A

[B]50

[C]50

K½Að1 þ aM½BÞ 1 þ M½B þ N½Cð1 þ bM½BÞ þ K½Að1 þ aM½BÞ

1 þ K½A þ N½C Mð1 þ aK½A þ bN½CÞ

1 þ M½B þ K½Að1 þ aM½BÞ Nð1 þ bM½BÞ

K½Að1 þ aM½B þ cL½CÞ 1 þ M½B þ L½C þ K½Að1 þ aM½B þ cL½CÞ

1 þ K½A þ L½Cð1 þ cK½AÞ Mð1 þ aK½AÞ

1 þ K½A þ M½Bð1 þ aK½AÞ Lð1 þ cK½AÞ

K ½Að1 þ aM½B þ bN½Cð1 þ acdM½BÞÞ 1 þ M½B þ N½Cð1 þ cM½BÞ þ K½Að1 þ aM½B þ bN½Cð1 þ acdM½BÞÞ

1 þ N½C þ K½Að1 þ bN½CÞ Mð1 þ cN½C þ aK½Að1 þ bcdN½CÞÞ

1 þ M½B þ K½Að1 þ aM½BÞ Nð1 þ cM½B þ bK½Að1 þ acdM½BÞÞ

Predicting Dose–Response Curve Behavior

Table 2 Binding Isotherms of the Reaction Schemes Presented in Figure 4

51

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52

Hall

The behaviors of these systems are rather simpler than those of the related two ligand systems with the homotropic interactions discussed previously. When determining the interaction between an allosteric modulator and an unlabeled orthosteric ligand in a binding assay (Fig. 4A), the behavior of interest is that of the competition curve in the presence and absence of the modulator (the effect of the modulator on the radioligand will already have been characterized). The behavior of this system is reasonably intuitive. Positive modulators (b > 1) cause a decrease in the midpoint of the inhibition curve (IC50) of C and negative modulators (b < 1) cause an increase. The effect of the modulator on the radioligand is a slight complicating factor. However, the behavior is still intuitive when considered in terms of the effects of the modulator on the afﬁnities of the two orthosteric ligands. For example, a compound that interacts negatively with the radioligand (a < 1) but neutrally with the competitor (b ¼ 1) will cause a decrease (leftward shift) in the IC50 of the competitor at constant radioligand concentration, due to the decrease it causes in the afﬁnity of the radioligand. The mathematical models derived from the reaction schemes in Figure 4B and C illustrate the differences in behavior between systems where the modulators interact with the same site or different sites. In this case, the principles involved are very similar to those encountered when looking at the effects of agonists acting at the same or different sites in functional systems. Indeed, the allosteric constants in models of allosteric interactions are closely analogous to agonist intrinsic efﬁcacies. This analogy is particularly clear when agonism is considered in terms of the two-state model (2–5) or ternary complex model of G-protein–coupled receptor (GPCR) activation and its elaborations (2–5,7,25–28). In these models, the parameters that represent agonist intrinsic efﬁcacies are also allosteric constants. In the case where the allosteric ligands compete, each modulator causes parallel rightward shifts in the titration curve of the other without affecting its maximal effect (unless the allosteric constants are equal). This is directly analogous to the effects of a partial agonist on concentration–response curves to a full agonist (29)—the partial agonist causes parallel rightward shifts of the full agonist concentration–response curve where the response is greater than the maximal effect of the partial agonist. In theory, the shifts in the titration curves could be subjected to Schild analysis (30) to determine the afﬁnity and allosteric constant (see part 4 of the Appendix). However, unless one of the allosteric ligands is neutrally cooperative, this would require the experiment to be performed at multiple radioligand concentrations to extract the two constants. Thus, in this case the Schild plot is perhaps more useful as a diagnostic rather than an analytical tool. By analogy with the mechanism proposed by Prosˇka and Tucˇek (11) for the interaction of alcuronium with orthosteric muscarinic ligands, a variant of Figure 4B has been proposed for competition between alcuronium and gallamine at their allosteric site on the muscarinic receptor (31). In this case, the orthosteric ligand

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cannot bind or dissociate when either allosteric ligand is bound (in Fig. 4B, neither reaction A þ RB $ ARB nor A þ RC $ ARC can occur). In the case where the allosteric modulators bind to distinct sites, the diagnostic features are saturability of effect and changes in the maximal effect of one modulator in the presence of the other. In fact, this model is directly analogous to the allosteric two-state model (32) and so, therefore, is its behavior. If the binding of A is deﬁned as the ‘‘functional response,’’ then the description of the functional effects of the parameters of the allosteric two-state model are also true of scheme 4C. Parameters a and b control the maximal effect of B alone and C alone on the binding of A; the product abd controls the maximal effect of B and C together on the binding of A. d therefore allows two neutrally cooperative ligands (a ¼ b ¼ 1) to affect the equilibrium binding of A when added together, if d 6¼ 1. Parameter c only affects the afﬁnities of B and C and so does not affect the binding afﬁnity of A directly; it simply changes the concentrations of B and C required to mediate their effects. A comparison of the behavior of Figure 4B and C in the presence of ligands with the same allosteric constants is shown in Figure 5. The interaction between a positive and a negative modulator has been chosen in this case simply because it makes the differences in the surface plots clearer. There is, of course, no requirement that the ligands in a three-ligand, two–binding site model are speciﬁc for one of the binding sites, e.g., a model in which tubocurarine could interact with both the allosteric site and the orthosteric site has been proposed to explain the interaction between tubocurarine, [3H]N-methylscopolamine, and a number of other ligands at muscarinic receptors (33). The most complicated binding models have been proposed for the interaction of ligands at GABAA receptors. These receptors are known to have a number of allosteric regulatory sites. The models were used to interpret the interactions between multiple ligands at fourligand binding sites (34,35) in an attempt to model the interaction between GABA and general anesthetics or neurosteroids and antiepileptic drugs at GABAA receptors in vivo. MODELING THE FUNCTIONAL EFFECTS OF ALLOSTERIC LIGANDS There have been far fewer models published that deal with the functional effects of allosteric modulators than there have been for binding reactions. Several authors have taken a pragmatic approach, ﬁtting a Hill equation to the data from a functional system and assuming that the change in agonist potency in the presence of the modulator is given by the factor ð1 þ M½BÞ=ð1 þ aM½BÞ (e.g., Refs. 10,21,22). Implicit in this approach is the assumption that the allosteric modulator changes the afﬁnity of the agonist without affecting its intrinsic efﬁcacy. This is not an unreasonable assumption in cases where the receptor reserve of the system is low and

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Hall

Figure 5 A comparison of the effects on orthosteric ligand binding of allosteric modulators that act at the same binding site with those of allosteric modulators binding at distinct sites. (A) The interaction between a positive modulator (B, a ¼ 10) and a negative modulator (C, c ¼ 0.01) acting at the same binding site. Other parameters were [A] ¼ 1/K, K ¼ M ¼ L ¼ 1. The asymptotes of any given curve are simply the effects of the given concentration of that compound in the absence of the other. The midpoint of each curve shifts indeﬁnitely to lower potency. (B) The interaction between a positive modulator (B, a ¼ 10) and a negative modulator (C, b ¼ 0.01) acting at distinct binding sites. Other parameters were [A] ¼ 1/K, K ¼ M ¼ N ¼ 1, c ¼ d ¼ 1. When high concentrations of both ligands are present, the maximal effect differs from that of either compound alone. The change in the midpoint of the titration curves is saturable in each case.

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the maximal response to the agonist is unchanged. Also, of course, a discrepancy between the binding and functional estimates of the cooperativity would indicate an extra interaction in the functional system. A major contribution to our understanding of the effects of allosteric modulators in functional systems was made by Ehlert (14). In this paper, he described the application of pharmacological null methods to the analysis of concentration–response data derived in the presence of an allosteric modulator. This has provided a powerful tool for the derivation of the afﬁnity of allosteric modulators in functional systems. The equivalent of the Gaddum equation for the interaction of an allosteric ligand with a highly efﬁcacious agonist is DR ¼

KA þ ½A=B KA þ ½A

where KA is the dissociation constant of the modulator and B is the product of the allosteric constant and the change in intrinsic efﬁcacy of the agonist caused by the modulator. This method has since been extended to allow pharmacological resultant analysis to be performed on allosteric compounds (36,37). An alternative to this classical analytical approach is to derive the model within the framework of a model of receptor activation. This was my approach when I investigated the effects of an allosteric modulator in the two-state model (Fig. 6) (32). This model had previously been proposed to explain the effects of positive allosteric modulators at GABAA (benzodiazepines) and A1 adenosine receptors but without a mathematical formulation (19,38). Since it is formulated at the level of receptor activation, this model does not allow quantitative analysis of concentration–response data unless the system has little or no receptor reserve, but it does provide further insight into the potential behaviors of allosteric interactions. The behavior of a number of experimental systems can be mimicked, at least qualitatively, with the allosteric two-state model [Hall (32); compare Ref. 32 with, for example, Refs. 39–42]. The model extends the predictions of the ternary complex model in Figure 1A for binding interactions, as it allows an interaction between the intrinsic efﬁcacies of the ligands as well as binding cooperativity. For example, the binding of two agonists acting at different sites on a receptor should exhibit positive cooperativity purely due to their ability to stabilize the ‘‘active’’ state of the receptor. Indeed, if this is not the case, a degree of negative binding cooperativity must also be postulated to mimic the behavior. Some of the functional predictions were, perhaps, not unexpected. For example, if the allosteric ligand is an antagonist and only affects binding, the antagonism is surmountable as well as saturable. An interesting feature of the model is that coagonism arises as a natural consequence of the activation cooperativity constant (d). When d > 1, two compounds that act as antagonists individually can induce a functional response in combination; indeed, if d > 1/ab the interaction between two inverse agonists can also integrate to agonism.

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Hall αθι K

R*G

εL

εN

RG

θK

ARG

θN

N

ζM

AR*G

εθι N αει L

βζη M

εζη N αγδθιλµ K ζ N R*BG βεη L ζθλ N

RBG

βγδζηλµ M γζλ M εζηθιλµ N AR*BG

αβδεηιµ L

ARBG

γθλ K

αK AR* R* αL βM βγδ M K R AR αγδ K R*B AR*B M βL γM αβδ L γK RB ARB

L

=

Figure 6 The cubic ternary complex model extended to include an allosteric modulator (now therefore a hypercubic quaternary complex model). The inner cube of the scheme is identical to that of the allosteric two-state model. To allow this, the allosteric constants are labeled differently (15). The receptor exists in an equilibrium between two states, inactive (R) and active (R), governed by the equilibrium constant L. A and B perturb this equilibrium and change the afﬁnity of the receptor for G. Both of these effects change the level of signaling compared to basal since the response in this model is quantiﬁed as the concentration of complexes containing both R and G. K, M, and N are the unconditional association constants of A, B, and G, respectively, and a, b, and e describe their respective effects on the R $ R equilibrium. c, f, and h are the allosteric constants describing the binding interactions of A with B, G with B, and G with A, respectively. d, g, and i are the allosteric constants describing the changes in the ability of the ligands to perturb the R $ R equilibrium when pairs of ligands (A and B, B and G, and A and G, respectively) are bound. k and l are the allosteric constants that describe the change in the binding afﬁnities or ability to perturb the R $ R equilibrium, respectively, when all three ligands are bound. Source: From Refs. 15 and 32.

A more complicated model of allosteric interactions based on the cubic ternary complex model of GPCR activation (Fig. 6) has since been proposed (15), although its predictions have not been fully explored. Also, Parmentier et al. (43) have proposed a model of allosteric modulation of family 3 GPCRs, which accounts for the conformational changes in the ligand-binding domain (open vs. closed states) and its separation from the effector domain in this receptor family. In this model, the effector domain has active and inactive conformations, whose interconversion is inﬂuenced by the conformational state of the ligand-binding domain. A characteristic

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that these models have in common with the allosteric two-state model is that they lack receptor reserve. That is, the midpoints of binding isotherms and activation curves are the same. Receptor density is simply a scaling factor and does not affect agonist or inverse agonist potency or intrinsic activity. In the case of the Christopoulos and Kenakin model (15), this is because a limiting case of the system has been used to simplify the derivation. Speciﬁcally, it has been assumed that, along with the soluble ligands, the G-protein is present in large excess over the receptor and therefore that the free and total G-protein concentrations are identical. Indeed, similar derivations from the entire family of ternary complex models of GPCR activation also lack receptor reserve. To complete this chapter, I will derive an expression for the other limiting case of allosterism in a ternary complex model, that in which the receptor is in excess of the G-protein. To do this, I will use the general form of a model of allosteric interactions based on the simpler ternary complex model of GPCR activation (44). In the case where ½RT ½GT , this model does exhibit the behaviors expected from receptor reserve and will serve as the motivation for deriving a model of allosteric interactions using the operational model approach. This will illustrate the conditions under which these two quite different approaches to modeling receptor behaviors intersect. The reaction scheme from which the model is derived is shown in Figure 7A. I provide an outline derivation of the equation for the activation curves below. The equilibrium association constants for the elementary reactions in Figure 7A are deﬁned in the usual way, e.g., K¼

½AR ½A½R

and acdK ¼

½ARBG ½A½RBG

The conservation of mass equations (assuming [A] and [B] are not limiting) are ½RT ¼ ½R þ ½AR þ ½RB þ ½RG þ ½ARG þ ½BRG þ ½ARB þ ½ARBG ½GT ¼ ½G þ ½RG þ ½ARG þ ½BRG þ ½ARBG By making the appropriate substitutions from the deﬁnitions of the association constants, these give ½RT ¼ ½Rð1 þ L½G þ M½Bð1 þ bL½GÞ þ K½Að1 þ aL½G þ cM½Bð1 þ abdL½GÞÞÞ ½GT ¼ ½G þ L½R½Gð1 þ aK½A þ bM½B þ abcdKM½A½BÞ

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Figure 7 (A) The ternary complex model of G-protein–coupled receptor activation extended to include an allosteric modulator (now in fact a quaternary complex model). Note the strong similarity with the allosteric two-state model (the inner cube of Fig. 6). K, L, and M are the unconditional afﬁnity constants of A, G, and B, respectively. a, b, and c are the allosteric constants describing the binding interaction between A and G, B and G, and A and B, respectively. d is the allosteric constant describing the change in the binding cooperativities when all three ligands are bound to the receptor. (B) A schematic representation of an operational model of an allosteric interaction. The binding reactions in the center of the scheme are identical to those in Figure 1A. However, in this case KA¼ [A][R]/[AR] and KB ¼ [B][R]/[BR] are the unconditional dissociation constants of A and B. c is the allosteric constant describing the binding cooperativity of A and B and is equal to a in Figure 1A. The dotted arrows represent hyperbolic transducer functions with the midpoints given on the arrows. Note, in contrast to the usual formulation of the operational model this model allows for constitutive receptor activity by allowing the unliganded receptor to interact with the signal transduction cascade.

Substitution for [R] in the expression for [G]T provides an expression for [G]T in terms of [R]T and [G], which can be rearranged to the following quadratic in [G]: ½Gð1 þ M½B þ K½Að1 þ cM½BÞ þ Lð½RT ½GT Þ ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞÞ þ L½G2 ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ ½GT ð1 þ M½B þ K½Að1 þ cM½BÞÞ ¼ 0

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The solution of this equation (after simplifying the product of two negatives) is pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b þ b2 þ 4ac ½G ¼ ð3Þ 2a where a ¼ Lð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ b ¼ 1 þ M½B þ K½Að1 þ cM½BÞ þ Lð½RT ½GT Þ ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ and c ¼ ½GT ð1 þ M½B þ K½Að1 þ cM½BÞÞ Note, only the positive square root can give a physically meaningful (nonnegative) solution to the quadratic. In the ternary complex model, the active species are the receptor– G-protein complexes, thus the response, E, is given by E ¼ ½RG þ ½ARG þ ½RBG þ ½ARBG which after making the appropriate substitutions from the equilibrium constant deﬁnitions gives E¼

L½G½RT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ 1 þ L½G þ M½Bð1 þ bL½GÞ þ K½Að1 þ aL½G þ cM½Bð1 þ abdL½GÞÞ ð4Þ

in which [G] is as deﬁned in Eq. (3). This is the most general form of the model and corresponds to that derived by Browning (44) but, given the form of [G], is far too complicated to allow derivation of useful expressions for the limits and midpoint in order to perform further analysis. It can, of course, be used to simulate concentration–response curve behavior at all concentrations of receptor and G-protein. The limiting cases of Eq. (3) are derived in part 5 of the Appendix. When ½RT ½GT , Eq. (3) simpliﬁes to ½G ½GT and E¼

L½GT ½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ 1þL½GT þM½Bð1þbL½GT ÞþK½Að1þaL½GT þcM½Bð1þabdL½GT ÞÞ ð5Þ

This expression can be derived much more simply by making the assumption that ½G ½GT initially, but does act as a useful check on the derivation of Eq. (3). Since the G-protein is in excess, this case of the model lacks receptor reserve; [R]T is only a scaling factor. If [R]T is varied,

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it changes the maximal effect that the system can generate but has no effect on (inverse) agonist potency or intrinsic activity. The allosteric constants in this model are directly analogous to those of the allosteric two-state model (I have used the same symbols for the allosteric constants in the current model to highlight this) and have the same effects on the behavior of the system as described by Hall (32). The concentration of G-protein controls the overall sensitivity of the system to agonism in this model since both the potency and the maximal effect of agonists and inverse agonists are hyperbolically related to [G]T. When ½RT ½GT , Eq. (3) simpliﬁes to ½G¼

½GT ð1þM½BþK½Að1þcM½BÞÞ 1þM½BþK½Að1þcM½BÞþL½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ

which after substitution into Eq. (4) and some manipulation (including use of the approximation ½RT þ½GT ½RT ) gives E¼

L½GT ½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ 1þL½RT þM½Bð1þbL½RT ÞþK½Að1þaL½RT þcM½Bð1þabdL½RT ÞÞ ð6Þ

This expression is very similar to Eq. (5), but with the role of [R]T and [G]T reversed. [G]T is now the scaling factor and the potency and intrinsic activity of agonists and inverse agonists are dependent on [R]T, i.e., the system can exhibit a receptor reserve [the limits and midpoint of Eq. (6) are summarized in Table 3]. The form and behavior of Eq. (6) suggest close parallels to an operational model of a similar system. Such a model is shown in Figure 7B. At the center of the ﬁgure is the reaction scheme showing a simple allosteric binding reaction (the same as in Fig. 1A). The equilibrium concentrations of the various receptor species are given by the following relationships (in this case using dissociation constants to be consistent with the operational model formalism). ½R ¼

KA KB ½RT KA KB þ KA ½B þ KB ½A þ c½A½B

½AR ¼

KB ½A½RT KA KB þ KA ½B þ KB ½A þ c½A½B

½BR ¼

KA ½B½RT KA KB þ KA ½B þ KB ½A þ c½A½B

½ARB ¼

c½A½B½RT KA KB þ KA ½B þ KB ½A þ c½A½B

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Equation

Parameter

(6)

Basal ([A] ¼ 0)

(6)

Midpoint

(6)

Maximum ([A] ! 1)

(7)

Basal ([A] ¼ 0)

(7)

Midpoint

(7)

Maximum ([A] ! 1)

General form

[B] ¼ 0

[B] ! 1

L½GT ½RT ð1 þ bM½BÞ 1 þ L½RT þ M½Bð1 þ bL½RT Þ

L½GT ½RT 1 þ L½RT

bL½GT ½RT 1 þ bL½RT

1 þ L½RT þ M½Bð1 þ bL½RT Þ Kð1 þ aL½RT þ cM½Bð1 þ abdL½RT ÞÞ

1 þ L½RT Kð1 þ aL½RT Þ

1 þ bL½RT cKð1 þ abdL½RT Þ

aL½GT ½RT ð1 þ bcdM½BÞ 1 þ aL½RT þ cM½Bð1 þ abdL½RT Þ

aL½GT ½RT 1 þ aL½RT

abdL½GT ½RT 1 þ abdL½RT

Emax vðbKB þ ½BÞ bKB ðv þ 1Þ þ ½Bðv þ bÞ

Emax v vþ1

Emax v vþb

adKA ðbKB ðv þ 1Þ þ ½Bðv þ bÞÞ bdKB ðv þ aÞ þ c½Bðv þ abdÞ

aKA ðv þ 1Þ vþa

adKA ðv þ bÞ cðv þ abd Þ

Emax vðbdKB þ c½BÞ bdKB ðv þ aÞ þ c½Bðv þ abdÞ

Emax v vþa

Emax v v þ abd

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Table 3 The Limits and Midpoint of Equations (6) and (7)

Note: Each is hyperbolic in [B]. The limits with respect to [B] are also shown.

61

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Let each of the species in the scheme interact with the transduction system in a manner that can be described by a rectangular hyperbola with the midpoint shown in Figure 7B. If we assume that each of the species must compete for a limited concentration of transducer proteins to result in a hyperbolic transducer function, then the equation of the response is E¼

Emax ðabd½R þ bd½AR þ ad½BR þ ½ARBÞ abdKe þ abd½R þ bd½AR þ ad½BR þ ½ARB

Substituting for the concentrations of the receptor species above and deﬁning v ¼ ½RT =Ke gives E¼

Emax vðadKA ðbKB þ½BÞþ½AðbdKB þc½BÞÞ adKA ðbKB ðvþ1Þþ½BðvþbÞÞþ½AðbdKB ðvþaÞþc½BðvþabdÞÞ

ð7Þ

I have used v for the ratio [R]T/Ke rather than s as would be the case in the operational model because it has a different signiﬁcance in this model—it deﬁnes the level of constitutive activity in and the overall sensitivity of the system to agonism rather than the efﬁcacy of individual agonists. Parameters a and b and the product abd denote the change in the midpoint of the transducer function when A, B, or both A and B are bound to the receptor. They are therefore a measure of the change in the strength of the stimulus provided by the receptor on ligand binding and can thus be deﬁned as intrinsic efﬁcacies. Parameter d is a cooperativity term [by analogy with d in Eq. (4)]: it deﬁnes the difference between the intrinsic efﬁcacy of the combination of both ligands (abd) and the combined intrinsic efﬁcacies of the individual ligands (ab). There is a close correspondence between the terms of Eq. (7) and those of Eq. (6); indeed, to translate between the two, it is simply necessary to make the following substitutions: Ke ¼ 1=L;v ¼ L½RT ; KA ¼ 1=K, KB ¼ 1=M; a ¼ 1=a; b ¼ 1=b; c ¼ c; d ¼ 1=d; and Emax ¼ ½GT . However, Eq. (7) is not simply a recasting of the ternary complex model equation. It applies to any system whose transducer function can be described by a rectangular hyperbolic function. The model of coagonism at N-methyl-D-aspartic acid receptors described by Corsi et al. (which was based on the more classical model of Marvizo´n and Baudry) is a more restricted operational model of allosteric interactions in the sense that the only active species is the ternary complex (ARB) (45,46). Not surprisingly, the behavior of Eqs. (6) and (7) is very similar to that of the allosteric two-state model. Thus, parameters a, b, c, and d of Eq. (6) correspond to the same parameters in the allosteric two-state model [as do their equivalents in Eq. (7)]. The major additional feature is the dependence of the effects of agonists and inverse agonists on receptor density. The effect of [R]T on the effect of allosteric inhibitors with different properties (b, c, or d<1) on the response to a high-efﬁcacy agonist is shown in Figure 8.

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From Figure 8, it is clear that an inhibitory effect that is mediated purely at the level of binding afﬁnity (c 6¼ 1) is insensitive to changes in [R]T while those that affect intrinsic efﬁcacy (b or d 6¼ 1) change their effect on the maximal response. When b 6¼ 1, effects on the basal activity of the system may also be seen. The limits and midpoint of Eq. (7) can be derived by comparison with Eq. (1) and are given in Table 3. I will now consider the results of potency

Figure 8 The effect of receptor density [[R]T ¼ 1 (diamonds), 10 (squares), or 100 (triangles)] on concentration–response curves to A in the absence (open symbols) and presence (closed symbols) of a maximally effective concentration of B in the models shown in Figure 7. Common parameters are L ¼ 0.0001 or Ke ¼ 10,000, K ¼ 1 or KA ¼ 1, M ¼ 1 or KB ¼ 1, a ¼ 100,000 or a ¼ 0.00001, [G]T ¼ 1 or Emax ¼ 1. (A) B is an allosteric inverse agonist. b ¼ 0.03, c ¼ 1, d ¼ 1 or b ¼ 100/3, c ¼ 1, d ¼ 1. (B) B is a negative allosteric modulator of binding. b ¼ 1, c ¼ 0.03, d ¼ 1 or b ¼ 1, c ¼ 0.03, d ¼ 1. (C) B is a negative allosteric modulator of receptor activation. b ¼ 1, c ¼ 1, d ¼ 0.03 or b ¼ 1, c ¼ 1, d ¼ 100/3.

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ratio (PR) and maximal effect ratio (MR) analysis (32) within this model. From Table 3, the potencies of the orthosteric agonist in the presence ([A]50) and absence ([A]50,[B]¼0) of the modulator are ½A50 ¼

adKA ðbKB ðv þ 1Þ þ ½Bðv þ bÞÞ aKA ðv þ 1Þ and ½A50;½B¼0 ¼ bdKB ðv þ aÞ þ c½Bðv þ abdÞ vþa

Thus, the PR is given by PR ¼ ¼

½A50 adKA ðbKB ðv þ 1Þ þ ½Bðv þ bÞÞ vþa ¼ ½A50;½B¼0 bdKB ðv þ aÞ þ c½Bðv þ abdÞ aKA ðv þ 1Þ bdKB ðv þ 1Þðv þ aÞ þ d½Bðv þ aÞðv þ bÞ bdKB ðv þ 1Þðv þ aÞ þ c½Bðv þ 1Þðv þ abdÞ

ð8Þ

Equation (8) is hyperbolic in [B]. When [B] ¼ 0, PR0 ¼ 1; when [B] ! 1, PR1 ¼

dðv þ aÞðv þ bÞ cðv þ 1Þðv þ abdÞ

ð9Þ

and the midpoint of Eq. (8) is PR50 ¼

bdKB ðv þ aÞ cðv þ abdÞ

ð10Þ

Also, from Table 3, the maximal response to the orthosteric agonist in the presence (Max) and absence (Max[B]¼0) of the modulator is as follows: Max ¼

Emax vðbdKB þ c½BÞ bdKB ðv þ aÞ þ c½Bðv þ abdÞ

and

Max½B¼0 ¼

Emax v vþa

Thus, the maximal effect ratio, MR, is MR ¼ ¼

Max Emax vðbdKB þ c½BÞ vþa ¼ Max½B¼0 bdKB ðv þ aÞ þ c½Bðv þ abdÞ Emax v bdKB ðv þ aÞ þ c½Bðv þ aÞ bdKB ðv þ aÞ þ c½Bðv þ abdÞ

ð11Þ

Again, this is hyperbolic in [B]. When [B] ¼ 0, MR0 ¼ 1; when [B] ! 1, MR1 ¼

vþa v þ abd

ð12Þ

and the midpoint of Eq. (11) is MR50 ¼

bdKB ðv þ aÞ cðv þ abdÞ

ð13Þ

Note PR50 ¼ MR50. As they stand, Eqs. (8) to (13) are not very helpful as they are composites of the intrinsic efﬁcacies of both ligands, the afﬁnity of the modulator, and v, a measure of the coupling efﬁciency of the system.

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However, some progress can be made by considering the following ratios: PR50 bdKB ðv þ aÞ cðv þ 1Þðv þ abdÞ bKB ðv þ 1Þ ¼ ¼ ½B50 ¼ PR1 cðv þ abdÞ dðv þ aÞðv þ bÞ vþb ð14Þ MR50 bdKB ðv þ aÞ ðv þ abdÞ bdKB ¼ ¼ MR1 cðv þ abdÞ ðv þ aÞ c

ð15Þ

This does not seem to have taken us very far, but we can now make an approximation that does allow the derivation of useful parameters. When the constitutive activity of the system is <10% of Emax, then v 1 and v þ 1 1. Thus, Eq. (14) reduces to PR50 bKB ¼ PR1 v þ b

ð14aÞ

Now, from Eq. (7), the maximal effect of B is MaxB ¼

Emax v vþb

Thus, the intrinsic activity (IAB) of B is IAB ¼ v=ðv þ bÞ (assuming that the response to a full agonist is Emax) and b=ðv þ bÞ ¼ 1 IAB . Thus, Eq. (14a) can be rewritten as PR50 ¼ KB ð1 IAB Þ PR1

ð14bÞ

Since intrinsic activity can be measured experimentally, Eq. (14b) allows the afﬁnity of B to be determined. Indeed, if the intrinsic activity of B is low ( < 0.1) the ratio PR50/PR1 is a good estimate of the afﬁnity of B without correction. Substitution of this estimate of KB into a rearrangement of Eq. (15) then yields MR50 bd ¼ KB MR1 c

ð16Þ

which is a measure of the overall cooperativity (both binding and activation) between A and B. In terms of Eq. (6) and Figure 7A, the left-hand side of Eq. (16) yields 1/bcd. Thus, by analyzing maximal effect and PRs in a functional assay with low constitutive activity it is possible to derive estimates of allosteric ligand afﬁnity and the overall cooperativity between the two ligands. This new method of analysis is illustrated in Figure 9. There are several points worth noting about this method of analysis. In the form given, it requires that the concentration–response curves have unit slopes. Preliminary exploration of systems with nonunit slopes using a Hill equation for the transducer function resulted in very unsatisfactory

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behavior. Indeed, the Hill equation is derived from a model of concerted binding reactions (e.g., nA þ R $ AnR) and excludes complexes containing nonidentical ligands. It is therefore unlikely to be an appropriate model for the integration of the signal from multiple receptor species. However, given concentration–response curves with unit slopes, the analysis applies to both positively and negatively cooperative interactions without modiﬁcation. Since PR50 should equal MR50, the analysis can be performed even if potency or maximal effect is left unchanged. Finally, note that the above analysis uses PRs rather than the equieffective concentration ratios of the null method (14). When the modulator affects the basal activity or maximal

Figure 9 (Caption on facing page)

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response to the orthosteric ligand, these two approaches can lead to different results. Indeed, if the modulator affects the basal or maximal response to the orthosteric ligand, the concentration–response curves are no longer parallel. It is then unclear which response level should be chosen for the null analysis as each will result in a different estimate of afﬁnity. SUMMARY The models that have been used to describe the binding interactions between allosteric ligands vary markedly in their complexity, from the simple ternary complex model (Fig. 1A) (13) to the hypercubic model of Van Rijn et al. (35). Indeed, there is no theoretical limit to the complexity of such models. The only limits are our ability to design experiments to test them and the application of Occam’s razor. Rather fewer models have been postulated for the functional behavior of allosteric interactions. Apart from Ehlert’s classical analytical treatment (14), these have generally been based on mechanistic models of receptor activation (15,32,43). In this chapter, I have presented an operational model of the functional behavior of allosteric systems. This model has the advantage of including the effects of receptor reserve and therefore allows predictions of the effect of changing

Figure 9 (Figure on facing page) An illustration of the analysis of potency and maximal effect ratios. Simulated data were generated from Eq. (7) to mimic experiments in which concentration–response curves to an orthosteric agonist were constructed in the presence of several concentrations of an insurmountable allosteric antagonist. A normally distributed random error with mean ¼ 0 and standard deviation ¼ 0.03 was applied to each simulated data point. Three replicate sets of data were generated. One of these is shown in (A). The concentrations of B were 0 (closed diamonds), KB (closed squares), 3KB (closed triangles), 10KB (closed circles), 30KB (open diamonds), and 100KB (open squares). Other parameters were: [R]T ¼ 10, v ¼ 0.001, KA ¼ 1, KB ¼ 1, a ¼ 0.00001, b ¼ 1, d ¼ 100, c ¼ 10, and Emax ¼ 1. The Hill equation was ﬁtted to the simulated curves. The midpoints and maximal effects of A derived from the curve ﬁts are shown in Table 4 along with the potency and maximal effect ratios. The potency ratio and maximal effect ratio curves for the data shown in (A) are given in (B) and (C), respectively. The Hill equation was ﬁtted to the data with the following constraints: the value at [B] ¼ 0 was constrained to unity; and the midpoints of the potency ratio and maximal effect ratio curves were forced to the same value (for a given data set). Unconstrained ﬁts showed the midpoints of the potency ratio and maximal effect ratio curves did not differ signiﬁcantly. Estimates of KB and the overall cooperativity (bd/c) between A and B were derived as described in the text [Eqs. (14b) and (16); Table 5]. The geometric mean of the estimates of KB was 0.88 and the geometric mean of the estimates of the overall cooperativity was 10.8. Both were close to their true values of 1 and 10, respectively.

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Table 4 Midpoints, Maximal Effects, Potency Ratios, and Maximal Effect Ratios of the Three Sets of Simulated Concentration–Response Data Referred to in Figure 9. Midpoint/ 103KA

Maximal effect/ Emax

Potency ratio

Maximal effect ratio

[B]

Set 1 Set 2 Set 3 Set 1 Set 2 Set 3 Set 1 Set 2 Set 3 Set 1 Set 2 Set 3

0 KB 3KB 10KB 30KB 100KB

8.3 16.4 20.6 30.2 37.8 38.5

9.7 17.6 20.8 40.9 50.5 45.5

11.6 16.3 30.5 37.5 56.1 44.2

1.048 0.846 0.735 0.660 0.527 0.508

0.982 0.893 0.702 0.687 0.554 0.452

0.948 0.885 0.742 0.655 0.537 0.494

1 1.98 2.48 3.63 4.56 4.64

1 1.82 2.15 4.23 5.23 4.71

1 1.41 2.64 3.24 4.85 3.83

1 0.81 0.70 0.63 0.50 0.48

1 0.91 0.71 0.70 0.56 0.46

1 0.93 0.78 0.69 0.57 0.52

Note: The values for the data presented in Figure 9 are from Set 1.

receptor density or coupling efﬁciency on the functional behavior of an allosteric system. It is based on the simplest model of an allosteric binding interaction (Fig. 1A); however, a similar approach could be taken with a more complicated binding model if the underlying behavior of the system were thought to be more complex. I have also presented an alternative approach to the analysis of functional data from allosteric systems that generate concentration–response curves with unit slopes. This gives afﬁnity estimates similar to those derived from the null analysis under conditions where both apply (Fig. 10) but may allow modulator afﬁnities to be derived in circumstances where the null method is not appropriate (Fig. 9).

Table 5 The Values of PR50, MR50, PR1, MR1 Derived from Fitting Hill Equations to the Data in Table 4 Parameter

Set 1

Set 2

Set 3

PR50 (¼MR50) PR1 MR1 KB (¼PR50/PR1) Cooperativity (¼MR50/KBMR1)

3.6 4.3 0.54 0.83 8.0

4.5 5.1 0.45 0.88 11.2

4.6 4.0 0.36 0.91 13.9

Note: The value at [B] ¼ 0 was constrained to unity and the values of PR50 and MR50 were forced to be the same. KB and overall cooperativity (bd/c) were derived as described in the text [Eqs. (14b) and (16)].

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Figure 10 Comparison of the null analysis (14) with analysis of potency and maximal effect ratios. Three simulated data sets were generated as described in the legend for Figure 9, but in this case for a surmountable allosteric antagonist. (A) The mean of the three simulated data sets; vertical bars show the standard deviation. The concentrations of B were 0 (closed diamonds), 3KB (closed squares), 10KB (closed triangles), 30KB (closed circles), 100KB (open diamonds), and 300KB (open squares). Other parameters were: [R]T ¼ 10, v ¼ 0.001, KA ¼ 1, KB ¼ 1, a ¼ 0.00001, b ¼ 1, d ¼ 1, c ¼ 0.03, and Emax ¼ 1. The Hill equation was ﬁtted to the simulated curves. The lines through the data are the mean of the ﬁtted curves. (B) The data in (A) analyzed by the null method. This analysis gave pKB ¼ 0.19 0.11 (mean standard deviation) and log(a) ¼ 1.62 0.11. (C) The data in (A) analyzed using potency ratios (in this case the maximal effect is unchanged so MR1 ¼ 1 and we must assume MR50 ¼ PR50). This analysis gave pKB ¼ 0.11 0.08 and log(bd/c) ¼ 1.63 0.11. Thus, the afﬁnity and cooperativity estimates derived by the two methods are very similar. Note Ehlert’s a is deﬁned in the reciprocal sense to bd/c and hence the appropriate comparison is between log(a) and log(bd/c).

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ACKNOWLEDGMENTS I would like to thank Mrs. Vikki Barrett and Drs. Christopher Browning, Richard Knowles, and Terry Kenakin for their helpful comments on the content of this chapter.

APPENDIX 1. Limits and midpoint of Eq. (1) As stated in the Introduction, models of pharmacological systems can frequently be expressed in the form y¼

a þ b½Li c þ d½Li

ð1Þ

In the absence of Li (i.e., [Li] ¼ 0), this expression simpliﬁes to y ¼ a/c. Note, in a binding reaction y ¼ 0 when [Li] ¼ 0, so a must equal zero for a binding isotherm. At saturating [Li] (i.e., when ½Li ! 1), y¼

a þ b½Li b½Li ! ¼ b=d c þ d½Li d½Li

The midpoint of Eq. (1) ([Li]50) occurs at the concentration of [Li] at which y is halfway between a/c and b/d, i.e., when 1 a b ad þ bc y¼ þ ¼ 2 c d 2cd Thus, ad þ bc a þ b½Li 50 ¼ 2cd c þ d½Li 50 ðad þ bcÞðc þ d½Li 50 Þ ¼ 2cd ða þ b½Li 50 Þ acd þ bc2 þ ad 2 ½Li 50 þ bcd½Li 50 ¼ 2acd þ 2bcd½Li 50 bc2 acd ¼ bcd½Li 50 ad 2 ½Li 50 cðbc adÞ ¼ d½Li 50 ðbc adÞ ½Li 50 ¼ c=d Proof that the Hill coefﬁcient of this curve is 1 is simply a question of deriving the equation of a Hill plot so I shall not reproduce it here. 2. Conditions for bell-shaped curves in Figure 1B If a titration curve is bell-shaped this implies that the binding equation has a local maximum somewhere within the range of [B] that was tested.

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In other words, there must be a point at which the ﬁrst partial derivative with respect to [B] of the binding equation is zero. For Figure 1B the binding isotherm is f ¼

K½Að1 þ aM½BÞ 1 þ ðM þ NÞ½B þ bMN½B2 þ K½Að1 þ aM½BÞ

where f is the fractional occupancy of A. The ﬁrst partial derivative of this expression with respect to [B] is @f aKM½A K ðM þ N Þ½A 2bKMN½A½B abKM 2 N½A½B2 ¼ @½B ð1 þ ðM þ NÞ½B þ bMN½B2 þ K½Að1 þ aM½BÞÞ2 This expression is equal to zero when the numerator equals zero, thus there is a turning point when aKM½A KðM þ NÞ½A 2bKMN½A½B abKM 2 N½A½B2 ¼ 0 Since [A] > 0, the factor K[A] can be cancelled from this expression to give ðM þ NÞ aM þ 2bMN½B þ abM 2 N½B2 ¼ 0 Thus, the local maximum occurs when qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2bMN 4b2 M 2 N 2 4ðabM 2 N ÞðM þ N aMÞ ½B ¼ 2abM 2 N qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 2bMN 4b2 M 2 N 2 4abM 3 N 4abM 2 N 2 þ 4a2 bM 3 N ¼ 2abM 2 N qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ bN þ b2 N 2 þ a2 bMN abðMN þ N 2 Þ ¼ abMN since only the positive root can give a value for [B] > 0. Now, for [B] to be a positive real number, the square root term must be greater than bN, i.e., b2 N 2 þ a2 bMN abðMN þ N 2 Þ > b2 N 2 from which the condition for the biphasic curves can be derived as follows: b2 N 2 þ a2 bMN abðMN þ N 2 Þ > b2 N 2 , aM ðM þ NÞ > 0 , aM > M þ N N ,a>1þ M

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3. Conditions for bell-shaped curves in Figure 1D As shown for Figure 1B above, we are looking for the criterion that forces a turning point to occur at a value of [B] > 0. For Figure 1D, the binding isotherm is f¼

ðK þLÞ½Aþ2cKL½A2 þðaKM þdLNÞ½A½B 2ð1þðK þLÞ½AþcKL½A2 þðM þNÞ½BþbMN½B2 þðaKM þdLNÞ½A½BÞ

The ﬁrst partial derivative of this expression with respect to [B] is ½AðbMNðS½B2 þ2ðT þ2cKL½AÞ½BÞþcKLðS½A2 þ2U½AÞþTU SÞ @f ¼ @½B 2ð1þT½AþcKL½A2 þU½BþbMN½B2 þS½A½BÞ2 where S¼aKM þdLN; T ¼K þL; andU ¼M þN. This derivative vanishes when its numerator equals zero. Thus, the [B] at which the maximum occurs is given by the solution of the quadratic pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b b2 4ac ½B¼ 2a where a ¼ bMNðaKM þ dLNÞ; b ¼ 2bMNðK þ L þ 2cKL½AÞ; and c ¼ cKL ðaKM þ dLNÞ½A2 þ 2cKLðM þ NÞ½A þ ðK þ LÞðM þ NÞ ðaKMþdLNÞ. Only the positive root can result in a positive value of [B] and this can only pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ be positive when 4ac > 0 (i.e., when b2 4ac > b). Now, a is made up of equilibrium constants and so is itself a positive constant for any given ligand. Thus, the factor 4a can be cancelled from this expression leaving the requirement that c > 0 or c < 0, that is, cKLðaKM þ dLNÞ½A2 þ 2cKLðM þ NÞ½A þ ðK þ LÞðM þ NÞ ðaKM þ dLNÞ < 0

ðA1Þ

pﬃﬃﬃﬃﬃﬃﬃﬃﬃ Now, when ½A ¼ 1= cKL inequality (A1) becomes cKLðaKM þdLNÞ 2cKLðM þNÞ pﬃﬃﬃﬃﬃﬃﬃﬃﬃ þ þðK þLÞðM þNÞðaKM þdLNÞ<0 cKL cKL pﬃﬃﬃﬃﬃﬃﬃﬃﬃ ,ðaKM þdLNÞþ2 cKLðM þNÞþðK þLÞðM þNÞðaKM þdLNÞ<0 pﬃﬃﬃﬃﬃﬃﬃﬃﬃ ,ðM þNÞ K þLþ2 cKL <0 pﬃﬃﬃﬃﬃﬃﬃﬃﬃ Since c, K, L, M, and N are all positive quantities ðM þNÞ ðK þLþ2 cKLÞ pﬃﬃﬃﬃﬃﬃﬃﬃﬃ cannot be negative, so bell-shaped curves cannot occur if ½A1= cKL.

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4. Schild analysis of the effects of competing allosteric ligands on orthosteric ligand binding. The analysis in this case gives the simplest expression when performed using IC50 ratios rather than concentrations yielding the same level of orthosteric ligand binding. For the reaction scheme in Figure 4B, the IC50 of B ([B]50) in the presence of C is given by: ½B50 ¼

1 þ K½A þ L½Cð1 þ cK½AÞ Mð1 þ aK½AÞ

(see Table 2). In the absence of C this simpliﬁes to ½B50;½C¼0 ¼

1 þ K½A Mð1 þ aK½AÞ

Thus, the ‘‘dose ratio’’ (DR) (actually a potency-ratio) is given by DR ¼

½B50 ½B50;½C¼0

¼

1 þ K½A þ L½Cð1 þ cK½AÞ Mð1 þ aK½AÞ Mð1 þ aK½AÞ 1 þ K½A

¼

1 þ K½A þ L½Cð1 þ cK½AÞ 1 þ K½A

¼1þ

L½Cð1 þ cK½AÞ 1 þ K½A

DR 1 ¼ L½C

1 þ cK½A 1 þ K½A

Taking logarithms, this gives logðDR 1Þ ¼ log½C þ log

Lð1 þ cK½AÞ 1 þ K½A

The ﬁnal expression is linear in log[C] with slope ¼ 1. Its intercept is the negative log of the IC50 of C ([C]50) in the absence of B (Table 2). Like the experiment itself, this plot is more useful as a diagnostic tool rather than for analysis. The linearity is diagnostic of competition; the value of the intercept ([C]50) can be determined from a much less complicated experiment. However, for a neutrally cooperative ligand (c ¼ 1) the intercept simpliﬁes to log L, allowing an estimate of the afﬁnity of C to be obtained directly.

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5. The limiting values of Eq. (3) When ½RT ½GT ; ½RT ½GT ½GT and term b of Eq. (3) simpliﬁes to b ¼ 1 þ M½B þ K½Að1 þ cM½BÞ L½GT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ This makes b2 þ 4ac a perfect square: b2 þ 4ac ¼ ð1 þ M½B þ K½Að1 þ cM½BÞ þL½GT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ

2

Thus, b þ

pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b2 þ 4ac ¼ 2L½GT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ ¼ 2a½GT

and hence, [G] ¼ [G]T When ½RT ½GT ; ½RT ½GT ½RT and term b of Eq. (3) simpliﬁes to b ¼ 1 þ M½B þ K½Að1 þ cM½BÞ þ L½RT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ In this case, b2 þ 4ac ¼ ð1 þ M½B þ K½Að1 þ cM½BÞ þ L½RT ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞÞ2 þ 4L½GT ð1 þ M½B þ K½Að1 þ cM½BÞÞ ð1 þ bM½B þ aK½Að1 þ bcdM½BÞÞ which is not a perfect square but its square root can be simpliﬁed by analogy with the standard approximation pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ y ð47Þ x2 þ y x þ 2x Consider Eq. (A2): y 2 y2 xþ ¼ x2 þ 2y þ 2 x x

ðA2Þ

If x > y then y2 =x2 y and ðx þ ðy=xÞÞ2 x2 þ 2y, which implies that if x>y pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ y x2 þ 2y x þ x

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Thus, when ½RT ½GT pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ b2 þ4ac ¼ 1þM½BþK½Að1þcM½BÞþL½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ 2L½GT ð1þM½BþK½Að1þcM½BÞÞð1þbM½BþaK½Að1þbcdM½BÞÞ þ 1þM½BþK½Að1þcM½BÞþL½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ After substitution into Eq. (3) and rearrangement, this gives ½G¼

½GT ð1þM½BþK½Að1þcM½BÞÞ 1þM½BþK½Að1þcM½BÞþL½RT ð1þbM½BþaK½Að1þbcdM½BÞÞ

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31. Prosˇka J, Tucˇek S. Competition between positive and negative allosteric effectors on muscarinic receptors. Mol Pharmacol 1995; 48:605–702. 32. Hall DA. Modeling the functional effects of allosteric modulators at pharmacological receptors: an extension of the two-state model of receptor activation. Mol Pharmacol 2000; 58:1412–1423. 33. Waelbroeck M. Identiﬁcation of drugs competing with d-tubocurarine for an allosteric site on cardiac muscarinic receptors. Mol Pharmacol 1994; 46:685–692. 34. van Rijn CM, Willens-van Bree E, Zwart JPC, Rodrigues de Miranda JF, Dirksen R. A molecular model for the synergic interaction between c-aminobutyric acid and general anaesthetics. Eur J Pharmacol 1999; 371:213–226. 35. van Rijn CM, Willens-van Bree E. A four-ligand hypercube model to quantify allosteric interactions within the GABAA receptor complex. Eur J Pharmacol 2004; 485:43–51. 36. Black JW, Gerskowitch VP, Leff P, Shankley NP. Analysis of competitive antagonism when this property occurs as part of a pharmacological resultant. Br J Pharmacol 1986; 89:547–555. 37. Christopoulos A, Mitchelson F. Application of an allosteric ternary complex model to the technique of pharmacological resultant analysis. J Pharm Pharmacol 1997:781–786. 38. Bruns RF, Fergus JH. Allosteric enhancement of adenosine A1 receptor binding and function by 2-amino-3-benzoylthiophenes. Mol Pharmacol 1990; 38:939–949. 39. Maksay G, Thompson SA, Wafford KA. Allosteric modulators affect the efﬁcacy of partial agonists for recombinant GABAA receptors. Br J Pharmacol 2000; 129:1794–1800. 40. O’Shea SM, Wong LC, Harrison NL. Propofol increases agonist efﬁcacy at the GABAA receptor. Brain Res 2000; 852:344–348. 41. Urwyler S, Mosbacher J, Lingenhoehl K, et al. Positive allosteric modulation of native and recombinant c-aminobutyric acidB receptors by 2,6-di-tert-butyl-4(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501. J Pharmacol Exp Ther 2001; 60:963–971. 42. Figler H, Olsson RA, Linden J. Allosteric enhancers of A1 adenosine receptors increase receptor-G-protein coupling and counteract guanine nucleotide effects on agonist binding. Mol Pharmacol 2003; 64:1557–1564. 43. Parmentier M-L, Pre´zeau L, Bockaert J, Pin J-P. A model of the functioning of family 3 GPCRs. Trends Pharmacol Sci 2002; 23:268–274. 44. Browning C. The effect of receptor density on the binding and fucntional properties of the human A1 adenosine receptor. PhD Thesis, National Institute for Medical Research, Mill Hill, London, 2003. 45. Corsi M, Fina P, Trist DG. Co-agonism in drug–receptor interaction: illustrated by the NMDA receptor. Trends Pharmacol Sci 1996; 17:220–222. 46. Marvizo´n J-C, Baudry M. Receptor activation by two agonists: analysis by non-linear regression and application to N-methyl-D-aspartate receptors. Anal Biochem 1993; 213:3–11. 47. Riggs DS. The Mathematical Approach to Physiological Problems. Baltimore, Maryland: The Williams and Wilkins Company, 1963.

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SECTION II: IONOTROPIC RECEPTORS

4 Allosteric Modulation of GABAA Receptors A Pharmacology of Sleep, Anxiety, Memory, and Consciousness Hanns Mo¨hler Department of Chemistry and Applied Biosciences, Institute of Pharmacology, University of Zurich, Federal Institute of Technology (ETH) and Collegium Helveticum, Zurich, Switzerland

INTRODUCTION Ever since the haem–haem interactions of hemoglobin were ﬁrst described (1), this remarkable phenomenon has evoked much interest, since proteins were found to be able to mediate indirect interactions between distinct speciﬁc binding sites. In their classic paper on a plausible model of allosteric proteins, Monod et al. (2) write: ‘‘By their very nature, these allosteric effects cannot be interpreted in terms of classical theories of enzyme action. It must be assumed that interactions are mediated by a molecular transition (allosteric transition) which is induced or stabilized in the protein when it binds an allosteric ligand.’’ The concept of allosteric binding sites has been recognized throughout biology but has nowhere found more practical applications than in pharmacology and in drug development. The allosteric nature of drug target interactions is not only a peculiarity on the molecular level but also has repercussions on all levels of biological organization, affecting the type of cellular responses, systems properties, and behavior.

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GABAA RECEPTORS AS ALLOSTERIC PROTEINS: BIDIRECTIONAL MODULATION The initial proposal that benzodiazepine tranquilizers produce their effects by selective enhancement of GABAergic synaptic transmission (3,4) left the mechanistic basis for this effect open. The recognition of speciﬁc high-afﬁnity binding sites of benzodiazepines, termed benzodiazepine receptors by Mo¨hler and Okada (5) and Squires and Breastrup (6), included the ﬁnding that these sites were different from the GABA binding sites. An interaction between these two sites became apparent by the ﬁnding that benzodiazepines enhanced the binding of GABA (7) and GABA was found to enhance benzodiazepine binding (8). The localization of both binding sites on a common protein complex became apparent from biochemical evidence. The ﬁrst GABAA receptor protein was identiﬁed (9) using 3H ﬂunitrazepam as a photoafﬁnity label of the benzodiazepine site—later termed a1 subunit. The attempt to solubilize and purify the photolabeled site resulted in the isolation of a functional protein complex that bound both GABA and benzodiazepines (10–12). These ﬁndings made clear that the benzodiazepine binding site is part of the GABAA receptor channel complex and functions as an allosteric modulatory site. The benzodiazepines were therefore termed positive allosteric modulators or agonists acting at the benzodiazepine site. This allosteric interaction was the molecular basis of the pharmacological and clinical effects of the classical benzodiazepines such as their sedative, anxiolytic, anticonvulsant, and muscle relaxant properties. In contrast, various other ligands of the benzodiazepine site including b-carbolines and certain benzodiazepines were equally bound with high afﬁnity and speciﬁcity, however, displayed in animals a proﬁle of activity that is the mirror image of the agonist proﬁle (13,14). In increasing doses they were arousing, proconvulsive, anxiogenic, convulsive, and spasmogenic. In electrophysiological and biochemical assays they depressed the effect of GABA by shifting the dose–response curve for GABA to the right and depressing the maximum. They, thus, behave like noncompetitive GABA antagonists at the GABAA receptor. These ligands were termed inverse agonists of the benzodiazepine binding site (Fig. 1) (15). The situation turned out to be even more complex when a number of structurally very different compounds were also found to bind to the GABAA receptor at sites distinct from both the GABA binding site and the benzodiazepine binding site and to modulate the function of the receptor in both qualitatively and quantitatively different ways, acting as positive or negative allosteric modulators (Fig. 2). The ﬁnding that a receptor can mediate two opposite effects (which are blocked by the same antagonist (17) was initially met with reluctance, although theoretical considerations had predicted that such a situation would be found (18). The discovery of inverse agonism at the benzodiazepine site was the ﬁrst example of this bimodal functional regulation of a receptor

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Figure 1 Schematic representation of the bidirectional shift of the GABA dose– response curve through allosteric modulation of GABAA receptors via the benzodiazepine site. The direction of the shift depends on whether an agonist (diazepam) or an inverse agonist interacts with the benzodiazepine binding site. The nature of the allosteric interaction determines the lack of drug efﬁcacy in the absence of GABA. In addition, a ceiling effect of the agonist action is apparent, i.e., the agonist response does not, practically, surpass the physiological maximum obtained by GABA alone. For details see text.

and had a general impact on the views of the molecular aspects of ligand– receptor interactions. SYNAPTIC MECHANISM OF ALLOSTERIC ACTION AT GABAA RECEPTORS The allosteric nature of benzodiazepine actions is reﬂected in their pharmacology (Fig. 1). The presence of a benzodiazepine remains silent in the absence of GABA. The drug action is therefore restricted to active GABAergic synapses. Thus, benzodiazepines display not only molecular speciﬁcity by acting exclusively via GABAA receptors, but also synaptic speciﬁcity. At low concentrations of GABA the cellular response to GABA is strongly enhanced by a therapeutically active benzodiazepine. However, the agonist becomes practically inactive when all GABA receptors are maximally activated by GABA. This ceiling effect of the drug action provides a high level of clinical safety. In synapses, GABAA receptors are activated by a brief, nonequilibrium exposure to high concentrations of GABA. Consistent with an increase in the afﬁnity of the receptors for GABA, therapeutically active benzodiazepines prolong the decay of spontaneous miniature inhibitory postsynaptic currents (mIPSCs). Similarly, the amplitude of an mIPSC is enhanced by a benzodiazepine agonist (19,20) suggesting that the druginduced increase of the afﬁnity for GABA resulted in the recruitment of more

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Figure 2 Illustration of some of the drug-binding sites at GABAA receptors. a1b2c2 subunits constitute the most prevalent receptor. For details see text. Source: From Ref. 16.

receptors for activation by GABA. However, in other neuronal systems the amplitude of the mIPSC remained unaltered by a benzodiazepine agonist (20–22), which has been interpreted to indicate that the release of a single quantum of GABA saturates all of the available GABAA receptors in the respective synapses, inducing a maximal peak response without further enhancement by the drug. Thus, the postsynaptic receptor occupancy by GABA appears to be cell- and synapse-speciﬁc, reﬂecting local differences in the number of receptors or the GABA concentration in the cleft. Accordingly, the inﬂuence of benzodiazepine agonists on the amplitude of mIPSC appears to vary with the operational conﬁguration of the GABAergic synapse (20). In summary, the enhancement of a GABAergic inhibitory response by a benzodiazepine agonist is based on the prolonged decay of the mIPSC and a potential increase of the mIPSC amplitude. But even if the peak mIPSC amplitude is not enhanced per se, the drug-induced prolongation of individual

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mIPSCs will be reﬂected in an increased peak amplitude of the compound inhibitory response caused by the summation of several miniature currents (21). PARTIAL BIDIRECTIONAL MODULATORS OF GABAA RECEPTORS Among the ligands of the benzodiazepine site, the partial agonists were considered to hold great promise as future anxiolytic agents that largely lack sedation and muscle relaxation and undesired liabilities such as tolerance and dependence upon chronic use. Partial agonists should not only lack certain effects of full agonists or produce less intensive effects but theory also predicts that they should act as antagonists of full agonists for those effects which they fail to produce themselves or produce only partially. Partial agonists require a higher fractional receptor occupancy than full agonists for a given fractional effect. This has been clearly demonstrated for the partial agonist bretazenil (Ro 16–6028) in electrophysiological, biochemical, and behavioral assays (23–26). Behaviorally, bretazenil displayed the proﬁle of a very potent anxiolytic and anticonvulsant compound with greatly reduced sedative and myorelaxant efﬁcacy, acting in fact as an antagonist of full agonists in the latter two effects. Furthermore, there was a much reduced interaction with the effect of ethanol, a greatly reduced liability for tolerance, physical dependence, and positive rewarding activity. The distinct pharmacological proﬁle of bretazenil was explained by a higher receptor reserve in neuronal circuits mediating anxiolytic activity compared to the circuits mediating sedative effects. Thus, at a given receptor occupancy bretazenil would act as an anxiolytic without sedation. Unexpectedly, in clinical studies, bretazenil induced sedative effects to a degree which precluded its use as a daytime anxiolytic in man. Possibly, a spectrum of drug effects which is based on differences in efﬁcacy and in receptor reserve has a poor predictability from animal experiments to humans. An interesting potential therapeutic action of partial inverse agonists was expected to be a mild increase of arousal and attention as well as an improvement of cognitive functions. Although such ﬁndings were made in animal studies, inverse partial agonists have not found further interest primarily due to concerns of proconvulsive and anxiogenic side effects. ANTAGONIST OF ALLOSTERIC MODULATION The action of benzodiazepine receptor antagonists is fully accounted for by the competitive interaction at the receptor with ligands that possess either positive or negative efﬁcacy (agonists) and obey the rules elaborated for all competitive antagonists at other receptors. In thermodynamic terms, the binding of antagonists to the benzodiazepine receptor differs clearly from that of agonists (27–29) as is also found for other receptors. Flumazenil, an imidazobenzodiazepinone (17), is the representative of this class of benzodiazepine ligands. It blocks all the effects of agonists and inverse

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agonists mediated by the benzodiazepine site. It is a very important tool in research and its availability in therapy makes the use of benzodiazepine receptor ligands even safer. A virtual lack of functional interaction of ﬂumazenil with the binding and gating of GABA makes ﬂumazenil an ideal ligand to label benzodiazepine receptors in vitro and in vivo, as its binding is not affected by the presence of GABA. The increasing use of ﬂumazenil (or its derivative Iomazenil) in positron emission tomography (PET) and singlephoton emission computed tomographic (SPECT) studies is not only useful in studies of drug kinetics at the site of the receptor to assess fractional receptor occupancy, but may shed light also on the alterations of the receptor in central nervous system (CNS) pathology. The hope that ﬂumazenil may be a probe for unmasking endogenous ligands of the benzodiazepine site has so far not been realized. Even if such an endogenous ligand existed and its action would be antagonized by ﬂumazenil it would be extremely difﬁcult to interpret the corresponding behavioral results. Flumazenil shares with many competitive antagonists at other receptors a very weak positive intrinsic efﬁcacy. Any interpretation concerning potential endogenous ligands (agonistic or inverse agonistic) are therefore likely to be ﬂawed. GABAA RECEPTOR SUBTYPES: A NEW ALLOSTERIC PHARMACOLOGY Molecular cloning of distinct GABAA receptor subunits led to the recognition of structurally and anatomically diverse GABAA receptor subtypes, the large majority containing the allosteric benzodiazepine binding site (30,31). The typical pentameric benzodiazepine sensitive receptor complex contains two a, two b, and one c-2 subunit. Distinct types of a-subunits (a1, a2, a3, a5) and of b-subunits (b1, b2, b3) determine distinct GABAA receptors (Fig. 3). Most importantly, classical benzodiazepines do not differentiate among GABAA receptor subtypes and act indiscriminately at all subtypes. However, the selective pattern of expression of GABAA receptor subtypes opened the possibility to modulate distinct neuronal circuits, provided novel ligands were found which displayed a differential interaction with GABAA receptor subtypes based on either afﬁnity of efﬁcacy (Table 1). Such agents would be expected to share with the classical benzodiazepines excellent overall tolerability, but display therapeutic indications that are more selective than those of the classical benzodiazepines. As a prerequisite, it had to be determined which pharmacological effects were mediated by distinct GABAA receptor subtypes. The dissection of the receptor pharmacology was achieved experimentally by generating four lines of point-mutated mice in which the receptors containing a1, a2, a3, or a5, respectively, had been rendered diazepam insensitive. A conserved histidine residue (H) in the drug binding domain was replaced by an arginine (R) as ﬁrst exempliﬁed for GABAA receptors containing the a1 subunit (35).

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Figure 3 Molecular architecture of a GABAergic synapse and the subunit repertoire of GABAA receptor subtypes.

Table 1 GABAA Receptor Subtypesa Subunits a1b2c2

a2b3c2 a3bnc2 a5b1,3c2

a4bnd

a4bnc a6bnd

a6b2,3c2

Localization

Pharmacology

Major subtype (60%), synaptic and extrasynaptic

Benzodiazepine-sensitive. Mediates sedative and anticonvulsant activity Minor subtype (15–20%), Benzodiazepine-sensitive. synaptic Mediates anxiolytic activity Minor subtype (10–15%) Benzodiazepine-sensitive. Pharmacology yet unclear Less than 5% of receptors, Benzodiazepine-sensitive. extrasynaptic (cerebral cortex, Mediates modulation of hippocampus, olfactory bulb) temporal and spatial memory Less than 5% of receptors, Insensitive to benzodiazepines. extrasynaptic Sensitive to low concentration of ethanol Less than 5% of receptors, Insensitive to benzodiazepines extrasynaptic Small population, extrasynaptic Insensitive to benzodiazepines. (only in cerebellum) Sensitive to low concentration of ethanol Less than 5% of receptors, Insensitive to benzodiazepines synaptic (only in cerebellum)

a

The term benzodiazepine refers to diazepam and structurally related agents in clinical use. Source: From Refs. 32–34.

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In the respective point-mutated mice the pharmacological action linked to the point-mutated receptor would be missing and thereby reveal the pharmacological relevance of the respective receptor in wild-type mice. Since the subunit composition and distribution of GABAA receptor subtypes is largely conserved between rodents and non-human primates the results were expected to be relevant for the human condition (36). Subtype-speciﬁc ligands are therefore expected to provide a new pharmacology of CNS disorders as outlined below for insomnias, anxiety disorders, and deﬁcits in memory and learning (37–39). ALLOSTERIC MODULATION OF SLEEP Frequently, sedation is taken as a surrogate marker for hypnotic activity. The sedative component of benzodiazepines, measured by the reduction of locomotor activity, has been attributed to neuronal circuits expressing a1GABAA receptors, the most prevalent receptor subtype in the brain. Mice in which the a1GABAA receptor had been rendered diazepam-insensitive by a point mutation [a1(H101R)] failed to be sedated by diazepam (35,40). Ligands with preferential afﬁnity for a1 receptors such as zolpidem or zaleplon are used as hypnotics. Similarly, the changes in the electroencephalographic (EEG) pattern induced by zolpidem in wild-type mice were almost exclusively mediated via a1GABAA receptors (41). Sedative tolerance involved also the a5 GABAA receptors (42). However, the changes in sleep architecture (suppression of REM sleep) and EEG-frequency proﬁles (reduction of slow-wave sleep, increase in fast b-frequencies) induced by classical benzodiazepines are largely due to effects mediated by receptors others than a1 (43). The enhancement of a2GABAA receptors by diazepam appears to have the most pronounced effect on the sleep EEG in wild-type mice. When the a2GABAA receptor was rendered diazepam-insensitive by a point mutation [a2(H101R)], the diazepam-induced suppression of d-waves, the increase in fast b-waves in non-REM sleep ( >16 Hz) and the diazepaminduced increase of h-waves in REM sleep were strongly attenuated (44). Thus, the hypnotic EEG ﬁngerprint of diazepam can be dissociated from its sedative action. Future hypnotics might target changes in the EEG pattern, which are characteristic of physiological sleep and thereby aim at improving sleep quality. For instance, the GABA-mimetic gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-0l hydrochloride; synonym THIP), which interacts preferentially with a4b3d GABAA receptors in vitro (45,46) was found to enhance slow-wave sleep in vivo (47,48). ALLOSTERIC ANXIOLYSIS Since a1GABAA receptors were found to mediate sedation (35), the anxiolytic activity of benzodiazepines was expected to reside in one or several of the remaining benzodiazepine-sensitive GABAA receptors (a2, a3, a5). Indeed,

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the benzodiazepine site ligand L-838417, which showed efﬁcacy at a2, a3, and a5 but not a1GABAA receptors, proved to be anxiolytic in wild-type rats (40). Similary, partial agonist 3-heteroaryl-2-pyridones of the benzodiazepine site with efﬁcacy at a2, a3, and a5 receptors, but not at a1 receptors, were found to show anxiolytic activity in rodents (49). However, it was the functional differentiation of GABAA receptors by knock in point mutations that identiﬁed the selective anxiolytic substrate. It was the a2- but not the a3- or a5GABAA receptor which mediated the anxiolytic activity of diazepan (50). In a2(H101R) mice, but not a3(H102R) or a5(H105R) mice, diazepam failed to induce anxiolytic activity (light–dark paradigm, elevated plus maze) (Table 2). Similarly, pyridones of the benzodiazepine site with efﬁcacy at a2, a3, and a5 receptors, but not a1 receptors, were found to show anxiogenic activity in rodents. It remains to be clariﬁed to what extent the a3GABAA receptor component does contribute to the anxiolytic activity of these ligands. An a3-selective inverse agonist was anxiogenic and proconvulsant in rodents (49). The agonist TP003 with selective efﬁcacy at a3 GABAA receptors was anxiolytic although only at high receptor occupancy (54). Since benzodiazepines exert anxiolysis at low receptor occupancy, the contribution of a3 receptors is unlikely to be of major relevance. Thus, the strategy to develop novel daytime anxiolytics, which are free of sedation, is clear (33,37,38,54). a2GABAA receptors by their preponderant localization on the axoninitial segment of principal cells in the cerebral cortex and hippocampus can control the output of these cells. In addition, a2 receptors are the only GABAA receptors found in the central nucleus of the amygdala, a key area for the control of emotions (55). Thus, by their strategic distribution in brain areas involved in anxiety responses, a2GABAA receptors are key substrates for anxiolytic drug action.

Table 2 Allosteric Pharmacology of Different GABAA Receptor Subtypes Target

Latest development

References

a1GABAA receptor Sedation benzodiazepine site agonist a2GABAA receptor Anxiolysis benzodiazepine site agonists

a1-selective ligands efﬁcacous clinically a2/a3-selective ligands, efﬁcacious preclinically

a5GABAA receptor Memory benzodiazepine enhancement site inverse agonist

a5-selective inverse agonist, efﬁcacious preclinically

Kopp et al. (41) Rudolph et al. (35) Rudolph et al. (35), McKernan et al. (40), Lo¨w et al. (50), Collins et al. (49) Crestani et al. (51), Collinson et al. (52), Chambers et al. (53)

Source: From Refs. 38, 39.

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ALLOSTERIC ENHANCEMENT OF LEARNING AND MEMORY Hippocampal pyramidal cells express various structurally diverse GABAA receptors in a domain-speciﬁc manner. While a1- and a2GABAA receptors are largely synaptic, a5GABAA receptors are located extrasynaptically at the base of the spines and on the adjacent shaft of the pyramidal cell dendrite. The a5GABAA receptors are therefore in a privileged position to modulate the excitatory input arising at the spines via N-methyl-D-aspartate (NMDA) receptors. Mice with a partial deﬁcit of a5GABAA receptors in the hippocampus showed an improved performance in trace fear conditioning, a hippocampus-dependent memory task (51). Similarly, in a mouse line in which a5GABAA receptors were deleted in the entire brain (37,52), an improved performance in the water maze model of spatial learning was observed. Likewise, a partial inverse agonist acting at a5GABAA receptors enhanced the performance of wild-type rats in the water maze test (Table 2) (53). It is striking that the behavioral consequences of an impairment of a5GABAA receptors are opposite to those of an NMDA receptor deﬁcit as shown in spatial and temporal associative memory tasks. While mice with a deﬁcit in hippocampal NMDA receptors show a deﬁcit in the formation of spatial and temporal memory (56,57), the mice with a partial deﬁcit of a5GABAA receptors in the hippocampus display an improvement in spatial and temporal memory performance. Thus, it appears that these two receptor systems play a complementary role in controlling signal transduction at the hippocampal principal cells (58). While the initial results with the a5-selective partial inverse agonist, described earlier, support a role in memory function, it has to be veriﬁed that such ligands do not interfere with other hippocampal functions such as sensorimotor gating.

ALLOSTERIC MODULATION OF CONSCIOUSNESS GABAA receptor subtypes have been found to mediate anaesthetic drug action. In pioneering studies using mutated recombinant receptors, Mihic et al. (58a) and Belelli et al. (58b) identiﬁed amino acid residues in the second and third transmembrane regions of the a and b subunit, which are critical for the action of general anaesthetics. Mutation of asparagine 265 in the b2 or b3 subunit essentially abolished the modulatory and direct of propofol and etomidate on recombinant GABAA receptors as well as of loreclezole, furosemide, and the non-steroidal antiinﬂammatory agent mefenamic acid. When the corresponding point mutation was introduced into the b3 subunit gene [b3(N265M)] of a mouse, the ability of etomidate and propofol to suppress a pain-indced motor response (hindlimb withdrawal reﬂex) was practically absent in the point-mutated mice (59). Thus, the immobilizing action of these drugs, which is indicative of surgical tolerance, is critically dependent on GABAA receptors containing the b3 subunit. In addition, the loss of the

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righting reﬂex induced by etomidate and propofol was signiﬁcantly reduced in time in the b3 (N265M) mice, indicating that the hypnotic action of these drugs is mediated in part by b3-containing GABAA receptors and in part by other targets, in particular b2-containing GABAA receptors (59). Thus, b3containing GABAA receptors are crucially important for the immobilizing action of etomidate and propofol, whereas the hypnotic action of etomidate and propofol is mediated in part by both b2- and b3-containing receptors (59). With these result, b3-containing GABAA receptors have been identiﬁed as mediators of surgical tolerance. The discovery of a molecular substrate for the modulation of consciousness is a major step forward in deﬁning the neuronal representation of conscious perception. CONCLUSIONS As an exquisite tool to analyse brain functions, the principle of allosteric modulation continues to advance the pharmacology of neurological and psychotic disorders. By their distinct distribution, GABAA receptor subtypes provide molecular targets for selective neuronal circuits. The allosteric modulation of GABAA receptor subtypes opens powerful new therapeutic opportunities. Drugs that target restricted neuronal circuits are expected to display fewer unwanted side effects than the non-selective benzodiazepine drugs presently in clinical use and provide therapeutic options beyond those of benzodiazepines. ACKNOWLEDGMENTS I wish to express my gratitude to my colleagues who contributed with their enthusiasm and expertise to the exploration of GABAA receptor structure and functions for more than two decades: D. Benke, F. Crestani, J-M. Frtischy, B. Lu¨scher, T. Okada, J.G. Richards, and U. Rudolph. REFERENCES 1. Bohr C. Theoretische Behandlung der quantitativen Verha¨ltnisse bei der Sauerstoffaufnahme des Ha¨moglobins. Zentr Physiol 1903; 23:682–688. 2. Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965; 12:88–118. 3. Haefely W, Kulcsar A, Mo¨hler H, Pieri L, Polc P, Schaffner R. Possible involvement of GABA in the central actions of benzodiazepines. Adv Biochem Psychopharmacol 1975; 14:131–151. 4. Costa E, Guidotti A, Mao CC. Evidence for the involvement of GABA in the action of benzodiazepines: studies on rat cerebellum. Adv Biochem Psychopharmacol 1975; 14:113–130. 5. Mo¨hler H, Okada T. Demonstration of benzodiazepine receptors in the central nervous system. Science 1977; 198:849–851.

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5 Allosteric Interactions at the NMDA Receptor Channel Complex Manolo Mugnaini Biology Department, Psychiatry Center of Excellence for Drug Discovery, GlaxoSmithKline Medicines Research Center, Verona, Italy

INTRODUCTION Historical Perspective Glutamic acid is the main excitatory neurotransmitter of the mammalian central nervous system (CNS) and mediates neurotransmission across most excitatory synapses (1). Soon after the earliest description of the marked excitatory action of L-glutamic acid on the general electrical activity of the mammalian cerebral cortex (2), it became evident that a variety of amino acids had a depolarizing effect on single neurons of the CNS (3), the most potent of which was the synthetic derivative of the D-form of the naturally occurring L-aspartic acid, N-methyl-D-aspartic acid (NMDA) (4). In the following decades, a huge number of electrophysiological and pharmacological studies, together with the development of selective antagonists, led to the unequivocal distinction of NMDA receptors, within the wide variety of glutamate receptors, as those selectively activated by NMDA (5). Indeed, the NMDA receptor was the ﬁrst class of glutamate-gated ion channels to be clearly identiﬁed, followed by a-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA) and kainate receptor classes. Overall, NMDA, AMPA, and kainate receptors, also termed ionotropic glutamate receptors (iGluRs), are responsible for signal transduction at the 93

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postsynaptic level in the vast majority of fast excitatory synapses of CNS. NMDA receptors, in particular, are ubiquitously distributed in the brain and play a fundamental role in normal CNS function. Their peculiar characteristics, like calcium (Ca2þ) permeability (6) and voltage-dependent blockade by magnesium (Mg2þ) ions (7), make NMDA receptors critical for important physiological mechanisms, like long-term potentiation (LTP) and synaptic plasticity (the mechanisms underlying learning and memory), as well as pathological events like excess elevation of intracellular Ca2þ, which is thought to be the cause of cell death in many neurodegenerative diseases (8,9). Competitive agonists and antagonists, i.e., compounds that activate or block NMDA receptors through direct interaction with the neurotransmitter binding site, have proved to be neurotoxic or to have strong side effects, respectively. For this reason, positive and negative allosteric modulators are preferred to restore NMDA receptor activity to physiological levels, without causing excitotoxicity or complete blockade of normal receptor functions. In the 1970s and 1980s, the NMDA receptor channel complex was extensively investigated in terms of pharmacology and electrophysiology, with the result of the discovery of many allosteric modulatory sites, the most important of which is the glycine binding site (Table 1) (10). In the 1990s, NMDA receptors were cloned, revealing the multiple subunit composition of these channels and their complex heterogeneity (11). Following a brief summary of the molecular biology of NMDA receptors (and some considerations about the concept of allosteric interactions for these proteins), the present chapter will deal with a description of the pharmacology and the structural determinants of the most important allosteric modulatory sites of the NMDA receptor channel complex, in the context of its molecular diversity. Finally, the therapeutic potential of compounds designed for selectively targeting the different modulatory sites of the NMDA receptor will be discussed. Molecular Biology of NMDA Receptors So far, three families of NMDA receptor (NR) subunits have been identiﬁed by molecular cloning: the NR1, which is composed of eight isoforms generated by alternative splicing of a single gene; the NR2, which contains four subunits (NR2A, NR2B, NR2C, and NR2D) encoded by four different genes; and the NR3 family, which is formed by two subunits (NR3A and NR3B) encoded by two genes (12–20). NR1 variants differ according to the presence or absence of three different amino acid cassettes: N1 (exon 5), a segment of 21 amino acids in the N-terminus domain, and C1 (exon 21) and C2 (exon 22), segments of 37 and 38 amino acids, respectively, in the C-terminus domain. In the present review, NR1 splice variants will be identiﬁed according to the nomenclature proposed by Durand et al. (21), in which three subscripts (one for every cassette) indicate the presence (1) or the

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Table 1 Allosteric Sites on NMDA Receptor Localization Allosteric site Glycine Polyamines (glycine independent) Polyamines (glycine dependent) Histamine Arachidonic acid Steroid positive modulatory site Adenosine triphosphate Zn2þ (very high afﬁnity, voltage independent) Polyamines (low glutamate concentration) Steroid negative modulatory site Zn2þ (high afﬁnity, voltage independent) Phenylethanolamines Felbamate Proton modulatory sites, primary Proton modulatory sites, secondary Redox modulatory site, primary Redox modulatory site, secondary

Effecta " "

Subunit

Domain LBD ATD (R2)

"

NR1 NR10XX/NR2B interface NR1

" " "

NR10XX, NR2B NR1, NR2A NR2A, NR2B

Not determined Not determined SMD1

" "

NR2A, NR2B NR10XX

Not determined Not determined

#

NR2B

Not determined

#

NR2

Not determined

#

NR2A

ATD

# # #

NR2B NR2B NR1, NR2

#

NR10XX, NR2A

ATD Not determined M3–S2 linker, M4–S2 linker ATD

"#

NR1

LBD

"#

NR10XX, NR2A

ATD

Not determined

a

", The allosteric site is responsible for an increase of NMDA receptor function; #, the allosteric site is responsible of a decrease of NMDA receptor function; " #, the allosteric site can either increase or decrease NMDA receptor function. Abbreviations: NMDA, N-methyl-D-aspartic acid; NR, NMDA receptor; ATD, amino terminal domain; LBD, ligand binding domain; SMD1, ﬁrst steroid modulatory domain; M3, third hydrophobic domain; M4, fourth hydrophobic domain; R2, second regulatory region; S2, second polypeptide segment.

absence (0) of a certain cassette, or if it is indeterminate (X). NR1011, for example, indicates the splice variant lacking the N1 cassette but containing both C1 and C2 cassettes, whereas NR11XX is used to indicate all splice variants containing the N1 cassette, independently of the presence or absence of C1 and C2 cassettes.

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NMDA receptor subunits share the same structural features of other iGluR subunits: a large extracellular N-terminus domain; four hydrophobic domains (M1, M2, M3, and M4); and an intracellular C-terminus domain (Fig. 1). Three of the four hydrophobic domains, M1, M3, and M4, are transmembrane spanning segments, whereas one, M2, makes a re-entrant loop within the membrane, which, together with the M2 regions of the other subunits forming the ion channel complex, lines the pore (22). Finally, NMDA subunits possess an additional extracellular amino acid segment located between M3 and M4 transmembrane spanning regions, and two intracellular domains, between M1 and M2 and between M2 and M3. The part of the protein composed of a portion of the large extracellular N-terminus domain preceeding M1 (a polypeptide segment of around 150 amino acids, termed S1) and a portion of the segment between M3 and M4 (another segment of around 170 amino acids, S2) has structural similarities with some bacterial periplasmic binding proteins such as lysine/ arginine/ornithine binding protein and glutamine binding protein (23). Similar to these proteins, this part of the NR subunits, termed ‘‘ligand binding domain’’ (LBD), has a bilobate structure, with two domains (S1 and S2) that, adjusting the amino acid substrate in the cleft, pass from an open to a closed conformation (24). The neurotransmitter glutamate binds to the LBD of the NR2 subunits (25–27), whereas glycine binds to the LBD of NR1 subunits (28–31). At present, it is not clear if the LBD of NR3 subunits, which has structural homology to the LBD of both NR1 and NR2 subunits, binds glutamate, glycine, or a yet unidentiﬁed ligand. The ﬁrst part (around 400 amino acids) of the large extracellular N-terminus domain has structural homology with bacterial leucine/ isoleucine/valine binding protein (32) and polyamine binding protein (33). Similar to these proteins, this amino terminal domain (ATD) has a bilobate structure, with two regulatory domains (R1 and R2) that facilitate the accommodation of the ligand in a clam shell–like feature. Many allosteric modulators of the NMDA receptor bind to the ATD, which is thought to translate binding of these agents into alterations of the LBD and/or stabilize speciﬁc receptor conformations, therefore changing NMDA receptor function (34). Recent molecular modeling studies suggest that while in the resting (closed) state of the NMDA receptor the LBD has an open conformation, while the ATD has a closed conformation. Upon binding of the agonist (glutamate and glycine), the LBD closes and the receptor passes to an open state (in which both the LBD and the ATD have a closed conformation). Conversely, the desensitized (closed) state of the receptor is characterized by an ATD in its open conformation (35). Homomeric assembling of any of the subunits (NR1, NR2, or NR3) does not lead to the formation of functional channels in mammalian cell lines. Homomeric expression of NR1 subunits in Xenopus oocytes gives

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Figure 1 Schematic representation of a tetrameric NMDA receptor composed of two NR1 subunits (only one subunit is shown), an NR2A and an NR2B subunit. The NR1 subunit lacks the amino terminal N1 cassette and contains both the C-terminal cassettes C1 and C2 and is therefore termed NR1011, according to the nomenclature proposed by Durand et al. (21). Every subunit contains a large extracellular N-terminus, four hydrophobic domains (M1, M2, M3, and M4), and an intracellular C-terminus tail. Three of the four hydrophobic domains, M1, M3, and M4, are transmembrane spanning segments, whereas one, M2, makes a reentrant loop within the membrane, which, together with the M2 regions of the other subunits, lines the pore. The extracellular portion of every subunit contains two bilobate structures: an LBD, with the two polypeptide segments S1 and S2, and an ATD, with two regulatory regions (R1 and R2). The neurotransmitter glutamate binds to the LBD of NR2A and NR2B subunits. The coagonist/positive allosteric modulator glycine binds to the LBD of the NR1 subunit. The negative allosteric modulators Zn2þ and ifenprodil bind to the ATD of the NR2A and NR2B subunits, respectively. To exert its positive allosteric modulation, PS binds to the SMD (SMD1, small grid), whereas to exert its glycine-independent positive allosteric modulation, spermine binds to a site located on the R2 regulatory domain of both NR1 and NR2B subunits. Abbreviations: NMDA, N-methyl-D-aspartic acid; NR, NMDA receptor; LBD, ligand binding domain; ATD, amino terminal domain; PS, pregnenolone sulfate; SMD, steroid modulatory domain.

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functional receptors, but it is not clear if this is due to the association of mammalian NR1 subunits with Xenopus glutamate receptor subunit XenU1 (36). However, the contemporary coexpression of at least a member of the NR1 family and a member of the NR2 family gives functional receptors with much bigger currents in both Xenopus oocytes and mammalian cell lines (37–39). When the NR3 subunit is present in the complex, the result is inhibition of channel function (40). Coexpression of NR1 and NR3 subunits leads to the formation of non-NMDA, glycine excitatory receptors (19), whereas the functional assembly of NR2 and NR3 receptors has never been reported. Similar to other iGluRs (41), NMDA receptors are thought to assemble with a preliminary step of family-speciﬁc subunit dimerization [to form, for example, (NR1)2 or (NR2A)2], followed by dimerization of these dimers, to form a ‘‘dimer-of-dimers’’ or tetrameric receptor [e.g., (NR1)2(NR2A)2 or (NR1)2(NR2B)2]. Recent studies strongly support this hypothesis (42–44), conﬁrming previous reports suggesting a tetrameric structure with two NR1 and two NR2 subunits in the same channel complex (45). A schematic representation of a tetrameric (NR1)2(NR2A) (NR2B) receptor is shown in Figure 1 (only one NR1 subunit is shown). The Concept of ‘‘Allosteric Interaction’’ for NMDA Receptors The term ‘‘allosteric’’ was used for the ﬁrst time by Monod et al. (46) to deﬁne, in enzymes, accessory sites topographically distinct from the substrate-binding site or ‘‘isosteric’’ site (the terms were derived from the ancient Greek words ‘‘a´llos’’ and ‘‘ı´sos,’’ which mean ‘‘other’’ and ‘‘equal,’’ respectively). Binding of a ligand to the allosteric site was able to induce a conformational change in the protein and modulate the binding of the substrate to the isosteric site, with the result of a modiﬁcation in enzymatic activity. The term was later introduced to deﬁne any binding site, on a receptor protein, that was able to modulate the binding properties of the primary ligand (e.g., a neurotransmitter or a hormone) to its binding site or ‘‘orthosteric’’ site (from the Greek ‘‘ortho´s,’’ ‘‘straight’’), with the result of a change in receptor activity (47,48). The ability of the allosteric site to modulate the binding properties of the orthosteric site was termed ‘‘allosteric interaction’’ or ‘‘allosteric modulation.’’ According to this deﬁnition, the term ‘‘allosteric’’ can be used also to describe interactions between multiple, identical sites present on the same receptor protein, a phenomenon better known as ‘‘cooperative interaction.’’ It is the case of many ligand-gated ion channels, for which more molecules of the same ligand bind to the same receptor and binding of one molecule to one site can affect the binding properties to the other site [as occurs for the two c-aminobutyric acid (GABA) molecules, which bind to the same GABAA receptor (49), or two acetylcholine (Ach) molecules, which bind to the same Ach receptor (50)]. In the present review, to avoid confusion,

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‘‘allosteric sites’’ are deﬁned as sites topographically and morphologically distinct from the primary ligand binding site(s), or ‘‘orthosteric site(s).’’ In parallel, the term ‘‘allosteric interaction’’ will be used to identify the interactions between allosteric and orthosteric sites and ‘‘cooperative interaction’’ or ‘‘cooperativity’’ to deﬁne the interactions between binding sites topographically distinct (they are either orthosteric or allosteric sites) but identical in terms of ligand speciﬁcity. The term ‘‘allosteric’’ has also been widely used in the literature to describe the properties of proteins that exist in an equilibrium mixture of different conformational states, corresponding to different pharmacological activities (51). An example of allosteric proteins are nicotinic Ach receptors, oligomers composed of ﬁve subunits that may shift, through so-called allosteric transitions, between resting, active, quickly desensitized, and slowly desensitized conformations (52). Every binding site of the receptor protein, irrespective of whether it is an orthosteric or an allosteric site, has different binding properties depending on the conformational state. A speciﬁc ligand might have a greater afﬁnity for one state rather than another and, therefore, increase the proportion of the protein in that state. To avoid confusion, in a recent review, Christopoulos and Kenakin (48) suggested the use of the term ‘‘receptor isomerization,’’ rather than ‘‘allosteric transition,’’ to describe the conversion of receptors between multiple conformations. The way in which a ligand, by binding to a preexisting major conformational state, can shift the equilibrium toward that conformation is called conformational selection, whereas the mechanism by which a ligand modiﬁes the conformation through the binding process, as occurs during the allosteric interactions and the cooperative interactions, is termed conformational induction. It is reasonable to think that in NMDA receptors, being ligand-gated ion channels composed of multiple protein subunits, all these phenomena (that is, allosteric interactions, cooperative interactions, and receptor isomerization) might coexist. Indeed, many allosteric sites have been identiﬁed on the NMDA receptor channel complex (Table 1). Moreover, two glutamate binding sites are present in a single NMDA receptor (53) and although initial studies seemed to suggest that the occupancy of one site does not affect the afﬁnity at the other site (54,55), the absence of cooperative interactions cannot be completely excluded. Finally, the presence of multiple closed and open states has been revealed by many authors (56–59). Further complication derives from the fact that glycine, similar to glutamate, is an essential requirement for NMDA receptor activation (60) and binds to the LBD rather than other structures of the NR1 subunit, so that many authors consider this substance a coagonist rather than an allosteric modulator, raising the query of which site can be considered as the othosteric site (the glutamate binding site, the glycine binding site, or both?). For the purpose of this review, considering that most NMDA receptors are contained in pathways in which glutamate has the role of the

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neurotransmitter (being released from the presynaptic terminals in an activity-dependent manner, while glycine is present in the extracellular ﬂuid at more constant levels), the glutamate (or NMDA) binding site will be considered as the orthosteric site and the glycine binding site as the allosteric site. In addition, the effect of all compounds modulating NMDA receptor function in a noncompetitive manner will be described. This can be broadly classiﬁed as (i) compounds binding to an allosteric site and changing NMDA receptor activity through a mechanism of conformational induction (i.e., proper allosteric interaction); (ii) compounds binding to an allosteric site and changing NMDA receptor activity with a mechanism of conformational selection; (iii) compounds changing NMDA receptor activity in an indirect way (i.e., affecting the levels or the activity of other biological factors, which in turn modulate NMDA receptor function); and (iv) compounds that change NMDA receptor function through yet unidentiﬁed routes. Substances like (þ)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d) cyclohepten-5,10-imine (MK801), which, rather than modulating NMDA receptor function, completely block the receptor activity through binding to a site deep within the channel, will not be considered in this review. Similarly, this chapter will not deal with the mechanisms of the voltagedependent block of NMDA receptors by Mg2þ, block by high concentrations of Ca2þ, glycine-independent desensitization, and Ca2þ-dependent inactivation. Also, modulation of NMDA receptor function by mechanisms of phosphorylation, dephosphorylation, and interaction with intracellular regulatory proteins will not be covered in this review.

ALLOSTERIC SITES OF NMDA RECEPTORS The Glycine Binding Site The exciting discovery that the classical inhibitory neurotransmitter, glycine, markedly increased the action of glutamate at the NMDA receptor was ﬁrst made by Johnson and Ascher in 1987 (61). In the following years, several functional studies [reviewed by Thomson (62)] demonstrated that glycine interacted with a distinct recognition site, later found to reside on NR1 subunits. The positive allosteric nature of this interaction was further supported by some receptor binding studies in which it was shown, by means of saturation and displacement experiments performed at equilibrium conditions, that glycine increased the afﬁnity of glutamate for its binding site on NMDA receptors (63,64) and that this effect was reciprocal, with glutamate increasing the afﬁnity of glycine (65,66). It became even more interesting when glycine–glutamate interactions were investigated by means of fast perfusion systems, which made feasible the direct measurement of activation and desensitization kinetics of the NMDA receptor channel complex. These techniques made possible the

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discovery of the so-called glycine-dependent desensitization, a timedependent loss of NMDA receptor function (in the continuous presence of glutamate or NMDA) that could be almost completely overcome by increasing glycine concentration (67). Glycine potentiation of NMDAevoked current was detected both during the initial (peak) response and at equilibrium conditions (i.e., during the steady-state response recorded after the onset of desensitization), therefore conﬁrming both previous functional studies reporting a glycine-induced increase in NMDA response at equilibrium conditions (60,61) and binding studies reporting an increase of NMDA or glutamate afﬁnity at equilibrium conditions (63,64). Glycine potentiation of the steady state was much larger than that of the peak response, with the result that, by increasing glycine concentration, the difference between the steady state and the peak response (in other words, desensitization) progressively diminished. This effect was found to depend on the ability of glycine to dramatically speed up the rate of the recovery from desensitization (67). Considering that the afﬁnity of glycine during the peak response was higher than during the steady state (68,69), these data suggest that the afﬁnity of glycine for the resting and open states of the NMDA receptor was higher than its afﬁnity for the desensitized state of the NMDA receptor and that glycine favored the nondesensitized states of the NMDA receptor for a mechanism of conformational selection. To explain glycine reduction of desensitization, some authors have hypothesized the existence of further NMDA receptor states: a glutamate(or NMDA-) unbound, closed conformation, with high afﬁnity for glycine, and a glutamate- (or NMDA)-bound, closed conformation with low afﬁnity for glycine, but in rapid equilibrium with an open (active) conformation (57). This theory was later supported by results from electrophysiological experiments on recombinant NR1/NR2A receptors, showing that glycine afﬁnity was higher in the absence of glutamate (70). The model described the loss of glycine afﬁnity upon glutamate binding in terms of negative allosteric interaction (or negative cooperativity, following the authors’ terminology) between the NMDA and the glycine binding sites, which might seem somewhat misleading in the light of the more common reports of positive allosteric interaction or coagonism between these two sites. A model considering the coexistence of major discrete quaternary transitions (i.e., receptor isomerization) between clear conformational states (e.g., resting state, open state, desensitized state) and, within each state, local reorganization at the subunit level more directly linked to fractional glycine and NMDA (or glutamate) binding (in other words, conformational induction) might help in interpreting these high-resolution electrophysiological data and reconciling them to more classical studies performed at equilibrium conditions. Unfortunately, how fractional binding of glycine and glutamate can affect quaternary organization at each conformational state is still a matter of debate.

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The fact that NMDA receptors are hetero-oligomers of four subunits and that the glutamate and the glycine binding sites reside on different subunits raises the question of the possible existence of distinct NMDA receptors with different glycine or glutamate afﬁnities, and/or glycine–glutamate allosteric interactions. Monaghan et al. (71) were the ﬁrst to hypothesize the presence of NMDA receptor subtypes of this kind in rat brain. These authors noticed the different regional distribution, in rat brain sections, of radiolabeled NMDA site agonists and antagonists and initially explained this difference with the possible existence of ‘‘agonist-’’ and ‘‘antagonistpreferring’’ NMDA receptor subtypes, i.e., NMDA receptors with a relatively higher afﬁnity for NMDA site agonists and antagonists, respectively. In the same study, the authors also noticed that glycine potentiation of [3H]glutamate binding was regionally different in rat brain sections (with striatum, for example, less sensitive than the cerebral cortex) and that glycine, while increasing the binding of NMDA site agonists, had the opposite effect on the binding of NMDA site antagonists. Following these observations, these authors (71) theorized the possibility that agonist- and antagonist-preferring NMDA receptor subtypes might have, in addition to (or as an alternative to) intrinsic differences in agonist and antagonist afﬁnities, also different glycine–glutamate allosteric interactions and therefore be differently regulated (in terms of binding at the NMDA site) by the endogenous glycine, which is difﬁcult to be properly washed away in brain sections. This fact could explain why, in regions like the septum and striatum, for the predominance of NMDA receptors with a more efﬁcient glycine allosteric modulation, there was relatively more NMDA-sensitive [3H]glutamate binding (because of a greater glycine-induced potentiation of binding) and relatively less binding of [3H]-3-(()-2-carboxypiperazin4-yl) propyl-1-phosphonic acid (CPP), an NMDA site antagonist (because of a greater glycine-induced inhibition of [3H]-CPP binding). On the contrary, in other regions like the thalamus and the cerebral cortex, possibly for a less efﬁcient glycine–glutamate allosteric interaction, there was a relatively lower [3H]glutamate binding (corresponding to a smaller glycineinduced potentiation) and relatively higher [3H]-CPP binding (corresponding to a smaller glycine-induced inhibition). These differences could not be explained in terms of regionally different concentrations of endogenous glycine, as revealed by microdialysis studies (72,73). The theory that the differentially distributed receptor subtypes revealed by Monaghan et al. (71) differed in the efﬁciency of the glycine– glutamate interaction was not demonstrated until the discovery of [3H]-D, L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP39653) (74). The high afﬁnity and selectivity of this NMDA site antagonist, as well as its interaction with a single binding site, made [3H]CGP39653 the radioligand of choice for detecting NMDA receptors in studies of receptor binding and autoradiography (64,75,76). It was therefore possible to determine that

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(i) glycine, while increasing the afﬁnity of glutamate, was able to decrease the afﬁnity of [3H]CGP39653; (ii) this inhibition was of allosteric nature, being reversed in a competitive fashion by selective glycine site antagonists, such as 7-chlorokinurenic acid (7-CKA) and 3-[2-(phenylaminocarbonyl) ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV150526A) (77,78); (iii) glycine inhibition of [3H]CGP39653 binding (similar to glycine enhancement of [3H]glutamate binding) was regionally different in rat brain sections (with striatum, for example, less sensitive than the cerebral cortex), suggesting the presence of regionally different NMDA receptor subtypes with different glycine–glutamate allosteric interactions; (iv) also, reversal of glycine inhibition by 7-CKA or GV150526A was regionally different in rat brain sections, but in a complementary fashion (with striatum, for example, more sensitive than the cerebral cortex), revealing the role of endogenous glycine in determining the different distribution patterns of NMDA site agonists and antagonists; (v) in brain membranes (in which the levels of endogenous glycine are much lower in comparison to brain sections) striatal and cerebral cortical NMDA receptors did not present signiﬁcant differences in terms of glycine or glutamate afﬁnity, or the afﬁnity of any other ligand used to demonstrate the existence of different allosteric interactions (Table 2); and (vi) the potency of glycine allosteric inhibition of [3H]CGP39653 binding was greater in the striatum than in the cerebral cortex (apparent pKi ¼ 7.48 and 6.98, respectively). In other words, the existence of the regionally distinct NMDA receptor subtypes reported previously was conﬁrmed (71) and it was proven

Table 2 Afﬁnity of Glycine and NMDA Site Ligands for Their Respective Binding Sites and Potency of Glycine in Glycine–Glutamate Allosteric Interaction, in the Striatal and Cerebral Cortical Membranes

Glycine binding site [3H]glycine 7-CKA (vs. [3H]glycine) GV150526A (vs. [3H]glycine) Glutamate binding site [3H]CGP39653 Glutamate (vs. [3H]CGP39653) Glycine–glutamate interaction Glycine (vs. [3H]CGP39653) a

Striatum

Cortex

pKD pKi pKi

7.08 0.08 (3) 6.89 0.09 (3) 8.47 0.10 (3)

7.01 0.04 (3) 6.71 0.07 (5) 8.49 0.02 (3)

pKD pKi

7.80 0.05 (4) 6.66 0.06 (3)

7.91 0.08 (8) 6.51 0.05 (5)

App pKi (high) 7.48a 0.05 (3) 6.98 0.02 (7) App pKi (low) 3.89 0.06 (3) 3.49 0.11 (7)

Signiﬁcantly different from the cerebral cortex. Abbreviations: NMDA, N-methyl-D-aspartic acid; 7-CKA, 7-chlorokinurenic acid; App, apparent. Source: From Refs. 64, 75, 76, and 78.

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that the difference between these receptor subtypes (at least for the striatum and the cerebral cortex) resided in the potency of glycine–glutamate allosteric interaction rather than in signiﬁcant differences in glycine or glutamate afﬁnity for their respective binding sites. It is difﬁcult to explain the features of these allosterically different NMDA receptor subtypes in terms of possible differences in subunit composition. In functional studies, the sensitivity of recombinant NR1/NR2A receptors to glycine is around 10-fold lower than that of NR1/NR2B, NR1/NR2C, and NR2D receptors (79–82). To some extent, binding studies also show similar results, with the afﬁnity of glycine for NR1/NR2A receptors being two to three times lower than that for NR1/NR2B receptors (83,84). Native NMDA receptors from the cerebral cortex and striatum, however, do not present signiﬁcant differences in terms of glycine afﬁnity, despite their relatively greater abundance in NR2A and NR2B subunits, respectively (Table 2). Following the ﬁnding that NMDA site antagonists and agonists have a slightly higher afﬁnity for NR1/NR2A and NR1/NR2B recombinant receptors, respectively (79,80), some authors have suggested that these subunit combinations might represent the antagonist- and agonist-preferring NMDA receptor subtypes previously found with receptor binding studies in native tissues (84,85). This result, however, was not conﬁrmed by other authors, who found a higher afﬁnity of both agonists and antagonists for the NR1/NR2A rather than the NR1/NR2B combination (25). In addition, receptor binding studies suggest that regions containing antagonist- and agonist-preferring NMDA receptor subtypes (namely, the cerebral cortex and the striatum) do not present signiﬁcant differences in terms of agonist and antagonist afﬁnities at the NMDA binding site (Table 2). Moreover, the relatively higher abundance of the NR2B mRNA in the striatum than the cerebral cortex (with respect to the NR2A) might suggest that this subunit confers agonist-preferring characteristics on NMDA receptors, but this is not true for the thalamus, which also contains relatively more NR2B than NR2A mRNA (14,86). Finally, the afﬁnity of [3H]glutamate was only slightly increased by glycine in the NR1/NR2B combination, when compared with the NR1/NR2A combination (84), which agrees with the smaller glycine-induced increase of [3H]glutamate binding and smaller glycineinduced decrease of [3H]CGP39653 binding in regions containing agonistpreferring receptors, like the striatum. This ﬁnding, however, does not explain why the same region presented the greatest enhancement of [3H]CGP39653 binding by 7-CKA or GV150526A. Interestingly, NR1/NR2A recombinant receptors appeared to form more efﬁciently than other combinations and looked very similar to native receptors in terms of binding properties (i.e., NMDA site agonist and antagonist afﬁnity) and allosteric interactions, with glycine increasing the afﬁnity of [3H]glutamate and inhibiting [3H]CGP39653 binding (25,84,87).

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Given the widespread distribution of the NR2A subunit mRNA in rat brain (which overlaps that of NR2B, NR2C, and NR2D subunits mRNA), it is reasonable to imagine that NR1/NR2A receptors may provide a core to which the other subunits can attach and inﬂuence the properties of the whole receptor. Indeed, the coexistence of more than one member of the NR2 family in the same receptor has been revealed by several authors (88–90). However, there are no reports of studies describing glycine– glutamate allosteric interactions in such complex combinations of NMDA receptor subunits (such as NR1/NR2A/NR2B, NR1/NR2A/NR2C, or NR1/NR2A/NR2D), so that a comparison with the NR1/NR2A combination and native receptors is not possible. In addition, different splice variants of the NR1 subunit family might account for the different glycine– glutamate allosteric interactions observed in native receptors. Glycine afﬁnity in native cerebellar NMDA receptors is lower than that of the cerebral cortex, especially at cerebellar granule cells (91). In addition, glycine did not potentiate NMDA-induced currents in cerebellar mRNA-injected oocytes, while, as expected, it potentiated cerebral mRNAinjected oocytes (92). These data might suggest that native NMDA receptors containing the NR2C subunit (which is highly expressed in the granular layer of cerebellum) have a lower afﬁnity for glycine and/or lower potency in the glycine–glutamate interactions. Functional and binding studies on heteromeric NR1/NR2 receptors, however, do not support this hypothesis, with glycine afﬁnity for NR1/NR2C receptors being similar or higher than that for NR1/NR2A and NR1/NR2B receptors (14,70,80,84). Glycine afﬁnity in recombinant NMDA receptors in a tri-heteromeric combination containing the NR2C subunit (e.g., NR1/NR2A/NR2C), however, has never been determined. The Zinc (Zn21) Binding Site Many experimental results have demonstrated that zinc ions (Zn2þ) inhibit NMDA receptor function by interaction with a speciﬁc site, distinct from the Mg2þ binding site and located on the extracellular surface of the receptor (93–96). In cultured neurons, Zn2þ inhibits NMDA receptor function in a voltage-independent manner, at concentrations as low as 1–10 mM, and in a voltage-dependent manner, at concentrations of 10–100 mM (97,98). These two effects of Zn2þ on NMDA receptor function have been termed as high-afﬁnity, voltage-independent inhibition and low-afﬁnity, voltagedependent inhibition, respectively. Among heteromeric NR1/NR2 receptors, the highest sensitivity for the high-afﬁnity, voltage-independent Zn2þ inhibition is given by those containing the NR2A subunit, suggesting the presence of a speciﬁc Zn2þ negative modulatory site responsible for this activity on this subunit (99–102). Indeed, more recent studies have revealed that Zn2þ resides in the cleft of

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the bilobate structure of the NR2A subunit (103–105) and that inhibition of Zn2þ is involved in the fast desensitization of NR1/NR2A receptors (106). Recent molecular modeling studies suggest that Zn2þ stabilizes the open state of the ATD, which corresponds to a desensitized form of the NMDA receptor (35). The sensitivity for the high-afﬁnity, voltage-independent Zn2þ inhibition of NR1/NR2A receptors is higher in the presence of protons (low pH), not only for the activation of the proton sensor located between the LBD and the transmembrane region (see section ‘‘The Proton Modulatory Site’’), but also for the protonation of histidine residues in the ATD, which per se increase the rate of formation of the desensitized state (35,104). The sensitivity of heteromeric NMDA receptors to voltage-independent Zn2þ inhibition is determined also by the presence of the N1 insert in the NR1 subunits, which, in fact, decreases both voltage-independent Zn2þ inhibition and proton (Hþ) inhibition of the NR1/NR2A and NR1/NR2B combinations, suggesting that Zn2þ and Hþ may share similar structural determinants (101,102). In line with this ﬁnding, glycine-independent spermine potentiation of NMDA receptor function relieves both voltage-independent Zn2þ and Hþ inhibition (102,108). The low-afﬁnity, voltage-dependent inhibition by Zn2þ is likely to occur within the ion channel pore and seems to involve the same residue on the M2 segment of the NR1 subunit implicated in Mg2þ block and Ca2þ permeability of the receptor channel (109–111). In addition to its well-known inhibitory effect at high concentrations, Zn2þ potentiates agonist-induced currents at submicromolar concentrations (EC50 ¼ 0.5 mM) in a voltage-independent manner (112). This effect, however, is seen only in homomeric receptors made of subunits lacking the N1 insert (NR10XX) and is absent in all kinds of NR1/NR2 heteromeric receptors, independently of the N1 insert in the NR1 subunit. The presence of native functional homomeric NMDA receptors, however, has never been demonstrated and the physiological relevance of the very high afﬁnity, voltage-independent stimulation of NMDA receptors is still to be resolved. The Phenylethanolamines Binding Site Ifenprodil, a phenylethanolamine derivative, is a noncompetitive NMDA site antagonist, initially thought to have antagonistic effect at the polyamine positive modulatory sites (113–115). In line with the glycine-dependent stimulatory effect of polyamines, which is present only with NR1/NR2B recombinant receptors, ifenprodil selectively inhibited NMDA receptors composed of the NR2B subunit, with a 400-fold higher afﬁnity for the NR1/ NR2B than for the NR1/NR2A combination (116,117). Later reports, however, suggested that ifenprodil acts on a site distinct from the polyamine binding site (118–120). In fact, residues on the NR2B subunit important

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for polyamine stimulation are not required for ifenprodil inhibition and vice versa. In addition, ifenprodil’s effect is independent of the presence of the N1 cassette in NR1 splice variants, unlike polyamine stimulation. Rather, polyamine and ifenprodil binding sites on the NR2B subunits seem to be allosterically linked in a negative manner (33,121,122). More recently, several mutagenesis studies have proved that the binding site of ifenprodil is located deep in the cleft of the ATD of the NR2B subunit (123–125). Functional studies suggest that ifenprodil (and structurally related compounds, like Ro 8–4304) antagonizes NMDA receptor function with an activity-dependent mechanism (126,127) and by increasing fast desensitization (106). Similar to the action of Zn2þ at the NR2A subunit, ifenprodil is thought to bind to the open state of the ATD domain of the NR2B subunit and stabilize the desensitized form of the NMDA receptor (35). As with Zn2þ inhibition, ifenprodil inhibition of NMDA receptor function is also potentiated by protons (128,129). However, mutation of several residues in the ATD of the NR1 subunit also affects sensitivity to ifenprodil, suggesting that some molecular determinants of the ifenprodil binding site may also be located on the NR1 subunits, or that these residues are important for the stabilization of the binding site or subunit–subunit interactions (124). Interestingly, neonatal rat brain NMDA receptors have a uniformly high afﬁnity for ifenprodil, which decreases during development because of the appearance of a lower afﬁnity component (117). This ﬁnding correlates well with the fact that NR2B subunits are expressed much earlier in development than NR2A subunits (130). The Polyamine Binding Sites Modulation of NMDA receptor activation by polyamines was ﬁrst reported by Ransom and Stec (131), who showed that spermine and spermidine increased the binding of [3H](þ)-5-methyl-10,11-dihydroxy-5H-dibenzo(a,d) cyclohepten-5,10-imine (MK801) to rat brain membranes, an index of NMDA receptor channel activation (132–134). In the following years, multiple, often opposing, effects of polyamines (i.e., stimulation and inhibition of NMDA receptor function) have been described and more than one interaction site has been hypothesized for this class of compounds (Table 1) (135–137). Nevertheless, the most relevant effect of spermine as an endogenous modulator remains its facilitatory inﬂuence on NMDA receptor– mediated neurotransmission (138), especially under pathological conditions such as brain ischemia, where its production is dramatically enhanced (139). The effect of polyamine on the afﬁnity of the primary neurotransmitter glutamate on native receptors is still uncertain, with studies reporting a small decrease of glutamate afﬁnity by 100 mM spermine (140) and higher concentrations of spermine (up to 1 mM) increasing the binding of

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[3H]glutamate (135). In contrast, receptor binding studies on native receptors clearly support the evidence that polyamines increase glycine afﬁnity to its binding site (65,141). Electrophysiological studies have proved the existence of two different mechanisms of positive modulation of NMDA receptor function by polyamines: so-called glycine-dependent and glycine-independent stimulation. Glycine-dependent stimulation of NMDA receptor function is directly linked to the spermine-induced increase of the afﬁnity for glycine at its modulatory site (see earlier paragraphs). Glycine-independent stimulation involves an increase of channel opening frequency and a decrease of the onset rate of glycine-independent desensitization (136,142–147). This effect, which can be revealed only at saturating concentrations of glycine, probably corresponds to the increase of [3H]MK801 binding observed in initial studies and is likely to be the most relevant effect of spermine on NMDA receptor function in physiological conditions. Interestingly, both glycine-dependent and glycine-independent stimulation are highly dependent on the type of NR1 splice variant and NR2 subunit (21). Glycine-independent stimulation by polyamines involves a relief of tonic Hþ and Zn2þ inhibition of NMDA receptor function (108) and is absent in NR1 splice variants carrying the N1 insert. This suggests that this segment, for its structural similarities to polyamines, might relieve NMDA receptors from tonic Hþ and Zn2þ inhibition by binding to the same site of polyamines. More recently, it has been shown that two residues critical for spermine potentiation are in the ATD of the NR1 subunit, very close to the site of the N1 insert, suggesting that this spermine binding site resides in the ATD of the NR1 subunit and that N1 insert constitutively occupies this binding site in NR1XX subunits. However, only receptors containing the NR2B subunit present glycine-independent stimulation by spermine, suggesting that this subunit also carries part of the binding site speciﬁc for the allosteric effect of polyamines (119,148,149). Recently, it has been proposed that spermine binds to residues between the R2 lobes of NR1 and NR2B subunits, stabilizing the dimer interface and the closed conformation of the ATD of these subunits, therefore favoring the open and resting states of the NMDA receptor and diminishing the number of receptors in the desensitized state (35). Another hypothetical site of spermine binding is the ﬁrst steroid modulatory domain (SMD1), another segment that may be involved in dimer formations (see section ‘‘The Steroid Modulatory Sites;’’ 150). In fact, it has been shown that glycine-independent spermine stimulation of NMDA receptor function is reduced by 80% if the SMD1 of the NR2B subunit is substituted with the corresponding sequence of the NR2D subunit, suggesting that SMD1 also plays a key role in this speciﬁc allosteric effect of polyamines. Finally, it is worthwhile to mention that Mg2þ ions, which block NMDA receptor channels in a voltage-dependent manner, at very high, millimolar concentrations, can potentiate NMDA receptor channels in a fashion similar to polyamines

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(i.e., in a voltage-independent and glycine-independent manner, only with NR1 splice variants lacking the N1 cassette and only with the NR1/NR2B combination), suggesting that Mg2þ may be the physiological agonist acting at the NR2B subunit–speciﬁc spermine site (151). Glycine-dependent stimulation of NMDA receptor function is seen only for homomeric NR1 channels and NMDA receptors carrying the NR2A and/or the NR2B subunits (21,33). Overall, these data are in line with the ﬁnding that the binding of [3H]MK801 to NMDA receptors in the cerebellum (which should contain a predominance of NMDA receptors carrying the NR2C subunits) appears to be less sensitive to the action of polyamines than is the case with forebrain receptors in which the NR2A and NR2B subunits are highly expressed (152,153). Electrophysiological studies on recombinant receptors have also conﬁrmed the existence of mechanisms of negative allosteric modulation by spermine, previously shown with receptor binding studies (see earlier paragraphs). Interestingly, in the presence of saturating concentrations of glycine, the magnitude of spermine stimulation was dependent on the concentration of glutamate (or NMDA) in the case of NR1/NR2B (but not NR1/NR2A) receptors: at low NMDA or glutamate concentration, spermine induced a small decrease in NMDA, and glutamate afﬁnity, which counteracted the stimulatory effect of spermine, resulting in little net effect of spermine (148). These results suggest that endogenous polyamines might act as a bidirectional gain control at some native NMDA receptors, by dampening the response at low concentrations of glutamate and enhancing the response at high concentrations of glutamate. Less relevant under physiological conditions is a mechanism of voltage-dependent inhibition of NMDA receptor function, which occurs only at hyperpolarized potentials and in the absence of extracellular Mg2þ, possibly representing a direct block of the ion channel by spermine (142,144,146,154). This effect was present at NR1/NR2A and NR1/NR2B receptors but was absent on NR1/NR2C (81). The Histamine Binding Site The ﬁnding that histamine can modulate currents gated by the NMDA receptor dates back to 1984 (155). Later, it was found that the potent enhancement by histamine of NMDA receptor-mediated currents in pyramidal cells was the result of a direct interaction with NMDA receptors (156). The stimulatory effect of histamine is similar, in some respects, to the glycine-independent stimulation by spermine: the ability of histamine to positively modulate NMDA receptor function is present only with NR1 subunits lacking the N1 cassette and in the presence of the NR2B subunit (148,157). This evidence and certain similarities in structure between histamine and spermine may suggest that these substances act on NMDA receptors through interaction with the same allosteric binding site. Nevertheless,

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the stimulatory effect of histamine, unlike that of spermine, is characterized by a rapidly developing increase in magnitude followed by marked and slow desensitization to a steady-state level, indicating that histamine acts at a novel recognition site distinct from that of spermine (157). At high concentrations (1 mM), histamine also causes a voltage-dependent inhibition of NMDA currents, also at receptors that are not sensitive to stimulation by histamine. The nature of this inhibition is still unknown. The Arachidonic Acid Binding Site Arachidonic acid, similar to nitric oxide (NO), is a small signaling molecule that diffuses readily through both ﬂuid and lipid phases and is generated in neurons in response to activation of NMDA receptors. In turn, arachidonic acid potentiates native NMDA receptor channels, by increasing the probability of channel opening, with no change in open channel current (158). As a consequence, a positive feedback mechanism of NMDA receptor function is based on the activation of phospholipase A2 by Ca2þ (which enters the cell through the NMDA receptor) and the consequent production of arachidonic acid, which in turn potentiates NMDA receptor currents (159). The stimulatory effect of arachidonic acid is observed even with a saturating effect of agonists at the glutamate and glycine binding sites of the NMDA receptor and is not due to activation of protein kinase C or conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives; in addition, it is independent from Mg2þ, Zn2þ, and polyamine binding sites. Finally, it has been shown that arachidonic acid binds to a 131 amino acid residue domain on the amino terminal of NR1 subunits, which has signiﬁcant homology with fatty acid–binding proteins (160). These results suggest that arachidonic acid binds to a distinct allosteric site on the NMDA receptor. The NMDA receptors containing the NR2A seem to be more sensitive to arachidonic acid than the NR2B subunit–containing channels (161). The Felbamate Binding Site Felbamate (FBM; 2-phenyl-1,3-propanediol dicarbamate) is a potent anticonvulsant used in the treatment of seizures associated with Lennox–Gastaut syndrome in children and complex partial seizures in adults. Initially found to decrease NMDA-induced neuronal injury (but not kainate-induced neuronal injury) in cultured cortical neurons (162) and to reduce NMDA receptor–mediated postsynaptic potentials in hippocampal slices (163), FBM was ﬁnally characterized as a noncompetitive NMDA receptor antagonist selective for receptors composed of NR1 and NR2B subunits (164,165). Similar to ifenprodil, FBM enhanced the afﬁnity of agonists at the glutamate binding site. However, FBM increases the binding of [3H]glycine to the NMDA receptor (166) and, unlike ifenprodil, pH did not affect its afﬁnity for the NR1/NR2B receptor (164). A point mutation on the

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NR2B subunit, which affects receptor sensitivity to ifenprodil, does not dramatically change the afﬁnity of FBM, suggesting that ifenprodil and FBM bind to different sites. It has been shown recently that FBM selectively binds to the activated and desensitized states of NMDA receptors, with the result that the inhibitory effect of FBM is stronger with both higher NMDA concentration and longer NMDA exposure (167). As a consequence of this usedependent inhibitory mechanism, FBM is thought to antagonize the excessive activation that occurs during seizure discharges and preserve normal neuronal ﬁring. In addition, FBM was shown to stabilize the desensitized form of the NMDA receptor also in the absence of glutamate, possibly helping to prevent the formation of excessive NMDA currents that may occur in the presence of an intense rise in glutamate concentrations. The Proton Modulatory Sites The NMDA receptor appears to be highly sensitive to changes in the microenvironment: NMDA receptor responses are selectively inhibited by protons (Hþ), with IC50 values close to physiological pH. This suggests the existence, on the NMDA receptor, of one or more negative modulatory sites for Hþ and implies that NMDA receptor channels are under tonic inhibition of around 50% at physiological pH (168,169). Many authors have demonstrated that the proton sensitivity of NMDA receptors is a point of convergence for the execution of many allosteric effects, including the high-afﬁnity, voltage-independent inhibition by Zn2þ (104,170), the negative modulation by phenylethanolamines (128,129), and the glycine-independent enhancement of NMDA receptor function by polyamines (see section ‘‘The Zinc Binding Sites,’’ ‘‘The Phenylethanolamines Binding Site,’’ and ‘‘The Polyamine Binding Sites.’’). Protons and spermine (polyamines) seem to comodulate neurotransmitter-induced gating through a mechanism of negative allosteric coupling, with spermine increasing receptor activity by decreasing proton inhibition. On the contrary, ifenprodil (phenylethanolamines) and Zn2þ inhibit NMDA receptors by enhancing proton inhibition (104). Recent scanned mutagenesis experiments of the NR1 subunit indicate that residues that control proton inhibition are localized in discrete regions, namely, in the extracellular end of M3 and the adjacent linker leading to the S2 portion of the glycine binding domain (M3–S2 linker) and in the linker between the S2 region and M4 (M4–S2 linker; 107). Interestingly, the M3–S2 linker partially overlaps the so-called ‘‘lurcher motif’’ (SYTANLAAF), a sequence conserved in all glutamate receptors that control receptor gating and channel opening, whereas the M4–S2 linker is downstream from this motif. Also the M3–S2 and M4–S2 linkers of the NR2 subunits present the molecular determinants for proton sensitivity; nevertheless, the NR2A, NR2B, and NR2D subunits present, in the M4–S2 linker, a histidine (His) residue that is absent in the NR2C subunit and is likely to be an

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essential determinant of the reduced pH sensitivity of NR1/NR2C receptors (108). Finally, it is worth pointing out that the M4–S2 linker partially overlaps the SMD1, recently discovered on the NR2B subunit (see section ‘‘The Steroid Modulatory Sites’’). Interestingly, SMD1 is a critical determinant of proton sensitivity in NR10XX/NR2B receptors (150). The SMD1 sequence is conserved between NR2A and NR2B, but signiﬁcant differences exist between NR2B (and NR2A) and NR2C and NR2D. Substituting the SMD1 sequence of the NR2B subunit with that of the NR2D subunit decreases the proton sensitivity of NR1/NR2B receptors, suggesting that NR2D (and NR2C) SMD1 domains lack part of the residues that confer proton sensitivity to the NR2B (and NR2A) subunit. Other mutations that affect (although to a lesser extent) the sensitivity to protons are found in the ATD domain and S1 region of the N1 subunit (107), conﬁrming previous ﬁndings that some molecular determinants of proton inhibition may be located near the N1 insert site or in the ATD (108,124). Interestingly, NR1 subunits carrying the N1 insert (NR11XX) are less sensitive to proton inhibition and need a higher Hþ concentration (lower pH) to produce the same inhibition of NMDA receptor function (pH ¼ 6.6–6.8 and pH ¼ 7.2–7.4 for NR11XX and NR10XX, respectively). In addition, speciﬁc for the NR2A subunit, the protonation of some histidine residues in the ATD seems to inhibit NMDA receptor function (and potentiate the inhibitory effect of Zn2þ), by increasing the rate of ATD opening and desensitized state formation (35,104). Overall, these data suggest that the so-called ‘‘proton sensor,’’ previously identiﬁed in the proximity of the N2 insert of the NR1 subunit, actually resides in the M3–S2 and M4–S2 linkers of all NR1 and NR2 subunits. However, other proton modulatory sites, capable of modifying NMDA receptor function, reside in the ATD of NR1 and NR2 subunits. The Steroid Modulatory Sites The neurosteroid pregnenolone sulfate (PS), one of the most abundant neurosteroids synthesized de novo in the nervous system, speciﬁcally potentiates the response of the NMDA receptor, while inhibiting GABA, glycine, and non-NMDA iGluRs (171–173). In addition, it was shown that a variety of sulfated steroids modulate the NMDA response in either a positive or a negative direction with a high degree of structural speciﬁcity. Surprisingly, the interaction between positive modulators, such as PS, and negative modulators, such as pregnanolone sulfate (3a5bS) and epipregnanolone sulfate (3b5bS) is noncompetitive, suggesting the existence of distinct steroid positive and negative allosteric sites on the NMDA receptor (174–176). Functional studies on recombinant NMDA receptors demonstrated that residues on the NR2 subunit are key determinants of modulation of PS and 3a5bS (177). Indeed, very recently, Jang et al. (150) have identiﬁed a 78-aa segment on the subunit NR2B that mediates the selective

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potentiation of PS on NMDA receptors, the so-called SMD1. Interestingly, SMD1 is conserved within the iGluR family and corresponds to the amino acid sequence involved in regulating the gating kinetics and binding of a positive modulator, cyclothiazide, to the AMPA receptor (178). The domain contains two a-helices, J and K, located in the S2 region of the bilobate structure of the glutamate binding site and the contiguous fourth transmembrane domain (M4). More precisely, J and K helices reside at the dimerization interface between two AMPA subunits, and binding of cyclothiazide promotes, by rearrangement of structures at this interface, dimer formation and alleviates desensitization. Molecular modeling, based on AMPA receptor structure, suggests that SDM1 of the NR2B subunit may contribute residues to a hydrophobic pocket capable of accommodating PS (150). Within the NR2 subunit family, SMD1 is highly conserved, but signiﬁcant differences exist. This reﬂects the fact that PS potentiates the response of recombinant receptors containing the NR2A and NR2B subunits, while it inhibits the response of receptors containing the NR2C and NR2D subunits (177). Modifying the amino acid sequence near the inner interface on NR2B to that of NR2D eliminates positive allosteric modulation by PS. SMD1 is not involved in the negative modulation by 3a5bS, but participates in controlling proton sensitivity [see Section ‘‘The Proton Modulatory Sites’’ (150)]. Mutated NR1/NR2B receptors, lacking the SMD1 insert (replaced with the corresponding region of the NR2D subunit), are less sensitive to tonic proton inhibition and need more protons (lower pH) to produce the same inhibition of NMDA receptor function (pH ¼ 6.6 and 7.5 for mutated and wild-type NR1/NR2B receptors, respectively). In addition, they lose spermine potentiation, which depends on the relief of tonic proton inhibition. Therefore, SMD1 is important for PS potentiation, proton inhibition, and spermine potentiation. It should be underlined, however, that PS stimulates NMDA receptor channels via a route independent of the proton sensor; rather, it involves the SMD1, although the proton sensor and SMD1 may share some common elements (the M4–S2 linker). This is proven by the fact that the potentiating effect of PS is abolished by the lack of SMD1. The ATP Modulatory Site Adenosine 5’-triphosphate (ATP) has both inhibitory and facilitating effects on NMDA receptor activity, depending on the ATP concentration. Inhibition of NMDA receptors by guanine nucleotides has been reported previously using radioligand binding assays (179,180). Indeed, recent electrophysiological experiments on native and recombinant heteromeric NR1/NR2A and NR1/NR2B (but not NR1/NR2C) receptors expressed in Xenopus laevis oocytes have revealed that, at a low concentration of glutamate, ATP and other nucleotides behave as competitive antagonists of

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the glutamate binding site (181,182). However, at concentrations of glutamate high enough to displace ATP from the NMDA binding site, NMDA current is potentiated in the presence of ATP, indicating that ATP binds as a positive allosteric modulator of NMDA receptor function. Interestingly, the afﬁnity of ATP for its modulatory site on the NMDA receptor is 15-fold lower than that for the glutamate binding site, suggesting that a high concentration of ATP is needed to produce its potentiating effect. As a consequence, potentiation of NMDA receptor function by ATP is revealed in the presence of high concentrations of both glutamate and ATP, whereas the inhibitory effect of ATP can be revealed only at low concentrations of both glutamate and ATP. If we consider that ATP might be co-released with glutamate into the synaptic cleft, the ﬁnal action of ATP may consist in focusing and enhancing the effects of glutamate at regions near the transmitter release sites (182). Finally, at some synapses in which zinc ions are also released into the synaptic cleft, potentiation of NMDA receptor function by ATP may be further increased by chelation of Zn2þ ions and relief of tonic Zn2þ inhibition of NMDA receptor function (see earlier paragraphs). Overall, the evidence that ATP can enhance NMDA receptor function is in line with the ﬁnding that ATP promotes the induction of LTP via a direct action on NMDA receptors, with no involvement of P2X or P2Y receptors (183). The Redox Modulatory Site Reducing agents like dithiothreitol (DTT) potentiate NMDA receptor channels, while oxidizing agents are inhibitory (184), suggesting the existence of a redox modulatory site in equilibrium between a fully reduced state (thiolate anion, RS) and an oxidized state (disulﬁde, RS–SR). The effect of DTT has two components, a reversible potentiation, which disappears spontaneously by washout of DTT, and a persistent (irreversible) potentiation, which is abolished only by an oxidizing agent (185). The reversible potentiation is present only in the NR1/NR2A combination, suggesting that it may be mediated by DTT chelation of Zn2þ, which selectively inhibits the function of NMDA receptors containing the NR2A subunit. The persistent potentiation relies on reduction of two cysteine residues located in the S2 extracellular loop region (between segments M3 and M4) of the NR1 subunit, which constitute the proper redox modulatory site of the NMDA receptor (186). These residues are located in the hinge of the cleft of NR1 and therefore affect the movement of the clam shell–like LBD. When the disulﬁde bond is reduced, the structure is more ﬂexible and the closure of S1 and S2 domains around glycine is facilitated (31). There is increasing evidence, however, that cysteine residues of the ATD of NR10XX and NR2A subunits also play a role in sensitivity to redox agents (187). These residues might modulate the ﬂexibility of the bilobate

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structure of the ATD, as occurs in the LBD. Alternatively, some authors have suggested the existence of cysteine bridge links between the R1 domains of different subunits (35). NO–producing agents also inhibit NMDA receptor channels, probably via donation of NOþ ions to the cysteine thiolate anions (RS) of the redox modulatory site of the NMDA receptor. This reaction, termed S-nitrosylation, leads to the formation of unstable S-nitrosothiols (RSNO), which more easily form disulﬁde bonds, resulting in a persistent block of the NMDA receptor channel (188–190). Interestingly, a negative feedback mechanism of NMDA receptor function has been proposed, which consists of stimulation of nitric oxide synthase (NOS) by calcium (which enters the cell following the opening of the NMDA receptor channel), with production of NO, which in turn inhibits NMDA receptor function via interaction with the redox modulatory site (159,188). OTHER SUBSTANCES MODULATING NMDA RECEPTOR FUNCTION In addition to the substances regulating NMDA receptors through the interaction with the allosteric modulatory sites described above, a variety of other compounds have been reported to modify NMDA receptor function through different (indirect) or still unknown mechanisms. This section will describe the effect of substances modulating NMDA receptor function through nonallosteric mechanisms, or for which a clear interaction with an allosteric site on the NMDA receptor has not yet been produced. Ethanol Ethanol inhibits NMDA receptor channels in a concentration-dependent manner (191–195). The sensitivity to ethanol seems to be independent of the NR1 splice variant, whereas NMDA receptors containing the NR2A and NR2B subunits are more sensitive to ethanol than those containing the NR2C subunit. Interestingly, ethanol sensitivity is enhanced by a calcium-dependent process that involves the interaction of the intracellular C0 domain of the NR1 subunit (a region of the cytoplasmic tail common to all NR1 splice variants) with proteins of the actin cytoskeleton (196). In addition, many reports suggest that ethanol inhibition of NMDA receptor function may occur through ethanol regulation of the activity of different protein kinases and phosphatases (which in turn change NMDA receptor function by phosphorylation or dephosphorylation) rather than through a direct interaction with an allosteric site on the NMDA receptor activation (197–200). A few studies, however, also suggest that ethanol may interact with speciﬁc amino acids in the M3 and M4 domains, which are involved in transducing agonist binding to channel opening and desensitization

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(201,202). In support of this theory, the kinetics of blockade of NMDA-gated currents by ethanol seem to be too rapid to be explained only in terms of phosphokinase activation (203). General Anesthetics Recent work has implicated NMDA receptor inhibition as a major source of CNS depression with general anesthetics, from gaseous anesthetics [e.g., nitrous oxide (N2O) and xenon (Xe)] to volatile anestethics (VAs; e.g., isoﬂurane) and intravenous anesthetics (e.g., ketamine; 204–208). Recently, it has been shown that the NMDA receptor is an essential requirement for the behavioral action of N2O (but not of VAs) in Caenorhabtidis elegans (209). Apart from dissociative anesthetics, like ketamine, which produce an open channel block of NMDA receptor function (like MK801), the molecular determinants of many anesthetics remain to be identiﬁed. Recent evidences, however, suggest that many different VAs produce a similar inhibition of NMDA-gated currents and that the kinetics for these agents are inconsistent with an open channel block or an effect mediated by phosphokinases; rather there is an interaction with an allosteric site (203). Interleukin-2 Interleukin-2 (IL-2) is a brain-derived glycoprotein that inﬂuences mesocorticolimbic dopamine release. Recently, it has been shown that, in voltageclamped neurons freshly isolated from the ventral tegmental area, IL-2, at physiologically relevant concentrations (0.01–10 ng/mL), inhibits NMDAinduced currents in a voltage-independent manner, while at higher doses (>50 ng/mL) it signiﬁcantly increases NMDA-induced currents in a voltage-dependent fashion (210). The inhibitory effect was competitive for the glutamate binding site of the NMDA receptor, whereas the obligatory requirement of intracellular ATP for the stimulatory effect suggests that this phenomenon may be determined by the ability of the neuron to maintain intracellular phosphorylation and therefore not be mediated by direct interaction of IL with an allosteric site on the NMDA receptor. THERAPEUTIC POTENTIAL OF ALLOSTERIC MODULATORS OF NMDA RECEPTORS Given their abundant and widespread distribution in mammalian neuronal tissue and their importance in excitatory transmission and normal CNS functioning, it is reasonable to imagine that NMDA receptors are involved in a variety of neuropsychiatric diseases and that drugs targeting this class of glutamate-gated ion channels may have great therapeutic potential. However, overstimulation of NMDA receptors, as occurs in the presence of high concentrations of glutamate or other competitive agonists, leads to excess

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intracellular calcium elevation and excitotoxicity (9). On the other hand, complete block of NMDA receptors, obtained in the presence of competitive NMDA site antagonists or channel blockers, may disrupt normal brain functioning and produce a series of adverse effects such as learning and memory impairment, neurotoxicity, disturbances of motor coordination, hallucinations, centrally mediated increase of blood pressure, catatonia, and anesthesia (211–218). Drugs acting at the different allosteric sites of the NMDA receptor may have the advantage of modulating NMDA receptor function without dramatically changing its basal activity; in addition, given the heterogeneity of allosteric modulations among recombinant receptor subtypes, they may selectively exploit their action through speciﬁc neuronal pathways. Positive Allosteric Modulators The hypothesis that hypofunction of the glutamatergic system might occur in schizophrenia was ﬁrst made by Carlsson and Carlsson (219), who noticed how NMDA channel blockers, like phencyclidine and ketamine, caused a schizophrenic-like syndrome in humans, recapitulating both the positive and the negative symptoms of this disease. In the following years, clinical and preclinical evidence strongly suggested that potentiation of NMDA receptor function may improve memory and cognition, and therefore be beneﬁcial in cognitive disorders and schizophrenia (220). More recently, it has been shown that enhancement of NMDA receptor function may facilitate extinction of conditioned fear, and therefore be beneﬁcial also in anxiety disorders (221). Glycine site agonists, or compounds acting through the steroid, polyamines, and ATP positive modulatory sites of the NMDA receptors may have beneﬁcial effects in treating these disorders. Indeed, glycine and glycine site agonists, like D-serine and D-cycloserine, have proven efﬁcacy when given in addition to standard antipsychotic therapy for the treatment of schizophrenia (222,223). Interestingly, overexpression of the NR2B subunit improves learning and memory in mice (224), suggesting that NR2B subunit–containing NMDA receptors may play a special role in the pathogenesis of this disease. In light of this hypothesis, targeting the site responsible for the glycine-independent stimulation of the NMDA receptor by spermine, located between the NR1 and the NR2B subunits, might be of special interest for treating schizophrenia. Negative Allosteric Modulators It is now generally accepted that cell death caused by sustained or prolonged NMDA receptor overactivation is the primary mechanism of neuronal death following cerebral ischemia (139) and may be an important cofactor of neuronal damage in many neurodegenerative diseases such as

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Parkinson’s, Huntington’s, and Alzheimer’s diseases (219,225–228). In addition, it is now well established that overactivity of excitatory pathways may be the source of epilepsy and neuropathic pain (229–232). There is also growing evidence that inhibition of NMDA receptor function may be beneﬁcial in counteracting different aspects of substance use disorders, from the expression and maintenance of morphine dependence (233,234), the acquisition of and relapse to cocaine addiction (235,236), and reverse tolerance to cocaine and amphetamine (237), to nicotine sensitization (238,239) and to ethanol withdrawal symptoms (240). Glycine site antagonists have proven to be efﬁcacious in several animal models of these diseases [see reviews (241–243)] without displaying the side effects reported for competitive NMDA site antagonists and NMDA channel blockers (244). Unfortunately, up to now, clinical trials with glycine antagonists have failed to meet preclinical expectations, showing little or no therapeutic beneﬁt (245–247). The difﬁculties associated with the interpretation of clinical data for complex pathologies like stroke and traumatic brain injury, together with the initial concern regarding brain penetration and brain availability of glycine antagonists, might have been the reason for these negative results. Hopefully, further trials with compounds with better pharmacokinetic proﬁles, supported by positron emission tomography studies monitoring the levels of NMDA receptor occupancy in healthy volunteers and patients, will reveal the potential therapeutic value of these compounds. NR2B subunit–selective negative allosteric modulators of NMDA receptor function can be obtained with compounds binding at the phenylethanolamine binding site (like ifenprodil). These compounds may have special interest, given the abundance of the NR2B subunit in the dorsal horn of the spinal cord and in the caudate putamen, for neuropathic pain and Parkinson’s disease, respectively. Indeed, there is signiﬁcant preclinical evidence of the antinociceptive and antiparkinsonian efﬁcacy of NR2Bselective compounds (248–250). Interestingly, mice deﬁcient in the NR2A subunit or the NR2C subunit show attenuation of focal ischemic brain injury (251,252), suggesting that NR2A-selective negative modulators, such as compounds acting through the high-afﬁnity Zn2þ binding site, or NR2C-selective inhibitors may be particularly efﬁcient neuroprotectants in cerebral stroke and similar pathologies. CONCLUDING REMARKS NMDA receptors are present ubiquitously in mammal CNS and are probably involved in a large variety of neurologic and psychiatric diseases. Their heteromeric structure, in terms of subunit composition and the large number of subunits cloned so far, suggests that many NMDA receptor subtypes may

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exist that play different roles in distinct neuronal pathways. The great number and heterogeneity of allosteric sites on NMDA receptors offer the potential to design drugs that selectively modulate the function of distinct NMDA receptor subtypes, or act on many NMDA receptors at the same time. Establishing the subunit composition of native NMDA receptors and their signiﬁcance in normal and disease states remains the major challenge at this point. Immunoprecipitation and puriﬁcation of native NMDA receptors, followed by their pharmacological characterization, may help in clarifying their subunit composition, whereas a more extended phenotypic characterization of knockout mice would be useful to understand the role played by different subunits in different pathologies. Especially the generation of conditional and tissue-restricted knockdown mice, with the use of RNA interference technology, would be of great help to study the function of speciﬁc NMDA receptor subunits and their involvement in the etiology of speciﬁc CNS diseases (253).

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6 5-HT3 Receptors Li Zhang Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland, U.S.A.

Sarah C. R. Lummis Department of Biochemistry, University of Cambridge, Cambridge, U.K.

INTRODUCTION The 5-HT3 receptor (previously also known as the serotonin type 3 receptor) is a member of the Cys-loop ligand-gated ion-channel family. It is therefore an allosteric protein and has many structural and functional similarities to the nicotinic-acetylcholine (nACh) receptor (1,2). 5-HT3 receptors mediate fast synaptic transmission through 5-HT-induced opening of transmembrane ion channels at synapses, and activation of 5-HT3 receptors in several brain regions can modulate the release of various neurotransmitters such as dopamine and glutamate (2–8). As this may underlie brain reward mechanisms, it is perhaps unsurprising that 5-HT3 receptors are one of the primary targets for the actions of alcohol and other drugs of abuse in the central nervous system (CNS) (9,10). Selective 5-HT3 receptor antagonists have been widely used for the treatment of emesis induced by chemotherapy and for irritable bowel syndrome (IBS) (11). In addition, there has been considerable research interest in 5-HT3 receptor antagonists for the treatment of a range of other disorders including addiction, anxiety, psychosis, nociception, and cognitive function (12–15). The original classiﬁcation of 5-HT receptors can be traced back more than half a century (16), but the true pharmacological and physiological identities of the 5-HT3 receptor have only emerged since the introduction 135

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of highly selective antagonists of 5-HT3 receptors in the mid-1980s. The subsequent cloning of complementary DNA (cDNA) encoding 5-HT3 receptor subunits greatly advanced our knowledge of 5-HT3 receptor pharmacology at the molecular level, and the structural organization of the ligand binding pocket has been the focus of much recent research and discussion. Data from other related ligand-gated ion channels (LGICs), and especially from nACh receptor studies, have shown which protein regions constitute the binding pocket, and also suggest that the basic structural details of all the receptors in this family will be similar (17). The crystal structure of the acetylcholine binding protein (AChBP), which is homologous to the extracellular domain of this family of proteins, has provided us with substantially higher resolution molecular details of the ligand binding domain, and has therefore provided a focus for the development of new highly speciﬁc and potent ligands (18,19). This chapter focuses on recent advances in the structural and functional characteristics of the 5-HT3 receptor, and its therapeutic potential in the central and peripheral nervous systems (PNS). 5-HT3 RECEPTOR PHARMACOLOGY There are currently many selective and potent compounds that act at this receptor. 5-HT3 agonists have in common a basic amine, an aromatic ring, a hydrophobic group, and two hydrogen bond acceptors, and their active compounds include 2-methyl-5-HT, phenylbiguanide and chlorophenylbiguanide (mCPBG), the latter of which is the most potent 5-HT3 agonist developed to date (20,21). Morphine and cocaine (16) were the ﬁrst antagonists used to characterize the 5-HT3 receptor, but using 5-HT as the origin, bemesitron and tropisetron were subsequently formulated. Later compounds that were developed include ondansetron, granisetron, and zacopride, which act at nanomolar concentrations, and there are now a wide range of similarly potent compounds. 5-HT3 receptor antagonists share a basic amine, a rigid aromatic or heteroaromatic ring system, and a carbonyl group (or isosteric equivalent) that is coplanar to the aromatic system (22,23), and here there are slightly longer distances between the aromatic and amine group when compared to the agonist pharmacophore. The 5-HT3 receptor can only accommodate small substituents on the charged amine, and a methyl group here appears to be optimal (23). Most of the potent antagonists of 5-HT3 receptors have 6,5 heterocyclic rings, and the most potent compounds contain an aromatic six-membered ring. The species differences in 5-HT3 receptor pharmacology have identiﬁed the roles of particular amino acids and/or regions of sequence in antagonist binding; for example, a number of residues in the C loop are strongly implicated to interact with D-tubocurarine. Docking of a range of antagonists into a model of the 5-HT3 receptor binding site shows reasonably good agreement with the pharmacophore model (24).

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RECEPTOR STRUCTURE The ﬁrst cDNA clone encoding a 5-HT3A receptor subunit, the mouse 5-HT3A receptor subunit, was isolated by functional screening of a mouse neuroblastoma (NCB20) cDNA library (1). Subsequently, the full-length cDNAs for orthologous 5-HT3A receptor subunits have been cloned from human (25), rat (26), guinea pig (27), and ferret (28). The amino acid sequence of the mouse 5-HT3A receptor subunit is homologous to other members of an LGIC superfamily that includes nACh, GABAA, and glycine receptors (1). Each subunit in this family consists of a large extracellular N-terminal domain, four transmembrane domains, an extracellular C-terminal domain and a large intracellular loop between TM3 and TM4 (Fig. 1). The predicted protein of the mouse 5-HT3A receptor subunit contains 487 amino acids and has a molecular weight of 55,966 (1).

Figure 1 Schematic representation of a 5-HT3A receptor subunit. It has a large extracellular domain on which are marked the approximate positions of the loops A–F, which are involved in forming the binding pocket (see also Fig. 2). The gray box represents the membrane, within which the four transmembrane spanning regions are located; of these the second (TM2) forms the channel pore. There is also a large intracellular loop between the third and fourth transmembrane spanning segments. Seven 5-HT3A receptor subunits have been cloned and conservation between the residues at the equivalent positions is shown in color.

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A high-resolution (X-ray crystal) structure is not yet available for the 5-HT3 receptor, but the AChBP, whose structure has been resolved to ˚ , has provided a good template to produce molecular models of 2.1 A the extracellular domain of this protein (18). Such a model reveals a curled b-sandwich structure in which the ligand binding sites lie at the interface of dimeric subunits (Fig. 2). The agonist and antagonist binding pocket is formed by three loops (A–C) from the ‘‘principal’’ subunit, and three (D–F) from the adjacent or ‘‘complementary’’ subunit. As in all Cys-loop receptors, the binding pocket contains a large proportion of aromatic residues, many of which are highly conserved and have roles in the binding of agonists or antagonists and/or in receptor gating. Table 1 lists key residues characterized as agonist/antagonist binding recognition sites located in each loop. The extracellular domain of the 5-HT3 receptor is linked via a network of multiple interacting loops to a transmembrane domain that consists of four transmembrane spanning segments (TM1–TM4). Combining these data with the model described above yields the structural model shown in Figure 2. Like other members of the LGIC superfamily, the TM2 region is thought to form the channel pore and has been extensively characterized by experiments involving site-directed mutagenesis and, in particular, the

Figure 2 A homology model of the extracellular and transmembrane domains of the 5-HT3A receptor showing the location of binding loops A–F. For clarity, only two of the ﬁve subunits are shown. The structure was created from a fusion of homology models based upon the crystal structure of AChBP (PDB ID; 1i9b) and cryo-electron microscopy of the nAChR (PDB ID; loed). Abbreviations: AChBP, acetylcholine binding protein; nAChR, nicotinic acetylcholine receptor.

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Table 1 Proposed Roles of Amino Acid Residues in the Extracellular Domain of the 5-HT3A Receptor Residue number W90109 W95109 W121110,114 P1 2 3109,114 N128110 E129111,112 F130109,110 Y141113–115 Y143113,115 G148113 E149113 V150113 Y153113–115 W183109,119 S227116,117 E231110 Y234114,115 E236118

Loop D D A A A A A E E E E E E B C C C C

AChBP equivalent W53 W58 W82 P84 Y89 N90 A91 L102 R104 G109 E110 V111 M105 W143 T185 C187 Y192 D194

Proposed role/location of the residue Agonist/antagonist binding site Receptor surface expression Receptor surface expression Antagonist binding site Agonist/antagonist binding site Antagonist binding site Antagonist binding site Antagonist binding site Antagonist binding site Antagonist binding site Antagonist binding site Antagonist binding site Binding and gating Agonist/antagonist binding site Antagonist-binding site Receptor trafﬁcking Antagonist binding site Agonist/antagonist binding site

Abbreviation: AChBP, nicotinic acetylcholine binding protein.

substituted cysteine scanning method (SCAM) (29,30). These studies have identiﬁed the residues that line the ion-accessible inner face of the channel pore and suggest that the channel gate is located in the center of TM2. As in other LGICs, the residues in the channel are predominantly nonpolar except for rings of charged amino acids. Further analysis has revealed that the ion selectivity of 5-HT3A receptor channels can be controlled by these charged rings, as changing these amino acids can convert this channel from cationic to nonselective or to anionic (31). The structure of the intracellular loop is the least understood part of the receptor. However, it does possess an amphipathic a-helical region containing a cluster of positively charged amino acids, which are involved in channel conductance (32,33). Substitution of these positively charged residues with noncharged equivalent residues from the 5-HT3B receptor subunit was found to increase the single-channel conductance of homomeric 5-HT3A receptors to the level of heteromeric receptors. Combining these data with structural information from the nACh receptor suggests that these charged residues line a series of ‘‘portals’’ that are the entry and/or exit route of ions to and from the channel (32,33). Thus both the TM2 region and the intracellular domain are critically involved in controlling ion ﬂow.

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RECEPTOR SUBTYPES Four classes of 5-HT3 receptor subunits have been identiﬁed since 1991, namely 5-HT3B, 5-HT3C, 5-HT3D, and 5-HT3E (34,35). 5-HT3B and 5-HT3C receptor subunits have 45% and 39% sequence identities with their 5-HT3A homologs, whereas the 5-HT3D and 5-HT3E subunits have identities of only 27% and 31%. Of all the ﬁve 5-HT3 subunits, only 5-HT3A receptor subunits are capable of forming functional homomeric complexes. These receptors, however, display anomalously low single-channel conductance when compared to some native 5-HT3 receptors (36), raising the possibility that heteromeric 5-HT3 receptors exist in vivo. Indeed coexpression of 5-HT3A with 5-HT3B subunits results in heteromeric complexes that have a singlechannel conductance similar to that of the native receptors described earlier (34). In addition to single-channel conductance, there are also several pharmacological and biophysical differences between homomeric (A) and heteromeric (AþB) receptors: heteromeric receptors are less sensitive to inhibition by D-tubocurarine (34) and picrotoxin (37), and differ in their modulation by certain anesthetics such as halothane (38). In addition, the 5-HT3AB receptors exhibit relatively low permeability to calcium ions (39), a linear-voltage relationship (34), and faster recovery from receptor desensitization (40). A recent study of the assembly of A- and B-subunits expressed in HEK293 cells using atomic force microscopy has suggested that the subunit stoichiometry is 2A:3B and the subunit arrangement is B–B–A–B–A (41). The functionality of the 5-HT3C, 5-HT3D, and 5-HT3E receptor subunits has not yet been determined.

DISTRIBUTION 5-HT3A receptor subunits have been found consistently in the CNS, and in the brain they have been identiﬁed in the cortex, hippocampus, nucleus accumbens, substantia nigra, and ventral tegmental area (42–44). High levels of the receptor are found in the brain stem, mainly in the nucleus tractus solitarius and area postrema (44). The localization of 5-HT3B receptor subunits in the CNS is currently controversial. Studies have reported that the signal for 5-HT3B receptors is detectable in human brain mRNA (34), and they have been located using immunolabeling in rat hippocampal neurons (45). However, studies with labeled riboprobes and reverse transcription polymerase chain reaction (RT-PCR) suggest that 5-HT3B subunits are restricted to the PNS (46). Thus, these subunits, if they are expressed in the CNS, could be in an extremely low abundance and/or in some small subpopulations of neurons. There is detectable signal for the expression of 5-HT3C but not 5-HT3D and 5-HT3E receptor subunits in the brain (35). The 5-HT3D and 5-HT3E receptor subunits appear to be restricted to the kidney, colon, and liver (35).

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In situ hybridization has demonstrated that 5-HT3A receptors are often colocalized with cholecystokinin mainly in GABAergic interneurons (47) and also with cannabinoid (CB1) receptors in neurons of the rat telencephalon, anterior olfactory nucleus, cortex, hippocampus, dentate gyrus, and amygdale (48,49). In these brain regions, 37–53% of all neurons expressing 5-HT3A receptors subunit also expressed CB1 transcripts, and 5-HT3A/ CB1-expressing neurons also contained GABA (49). These ﬁndings indicate possible interactions between CB1 and 5-HT3A receptors, and also suggest that these receptors may contribute to the regulation of GABA neurotransmission in the brain. At the subcellular level, there is strong evidence for differential localization of pre- and/or postsynaptic 5-HT3A receptors within different central regions, depending on the nature of the neurons containing 5-HT3A receptors (50). For instance, 5-HT3A receptor immunoreactivity is mostly abundant in postsynaptic dendrite sites in the hippocampus, whereas receptor immunoreactivity has been associated with presynaptic nerve endings in other brain areas such as the amygdala (44,50).

POSTTRANSLATIONAL MODIFICATIONS Modulation of 5-HT3 Receptor Function by Protein Kinases Topologically, the 5-HT3 receptor has a large cytoplasmic domain, which contains a cluster of putative protein-phosphorylation sites for various kinases (1). Activation of protein kinase A (PKA) substantially accelerates the desensitization kinetics of 5-HT3 receptors in mouse neuroblastoma cells and in HEK293 cells expressing 5-HT3 receptors (51,52), and phosphorylation has been observed on a putative PKA site in the cytoplasmic domain (53). The role of protein kinase C (PKC) is less clear: activation of PKC has been reported to increase the amplitude of 5-HT-activated current in heterologously expressed 5-HT3A receptors and in N1E-115 cells (54,55), but point etc. mutations of putative PKC sites did not affect the sensitivity of the mutant receptors to PKC potentiation (55,56). Modulation of 5-HT3 Receptor Trafficking by Intracellular Signaling Pathways The 5-HT3 receptor is the ﬁrst LGIC to have been tracked in real time from ‘‘birth’’ (receptor biogenesis) to ‘‘death’’ (reabsorption of receptors into the cell). These data, which use ﬂuorescence images of 5-HT3A receptors, have shown the dynamic processes of intracellular and membrane receptor trafﬁcking in transfected HEK293 cells (57). Intracellular 5-HT3A receptors were shown to trafﬁc to the cell surface at a velocity of less than 1 mm/sec, and could internalize within minutes after exposure to a selective 5-HT3 receptor agonist. Conversely 5-HT3B receptors are retained and degraded

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within the endoplasmic reticulum, unless they are ‘‘rescued’’ by coexpression with 5-HT3A subunits, which will effectively ‘‘carry’’ them to the cell surface. These processes can be modiﬁed by intracellular signaling pathways: activation of PKC by phorbol 12-myristate 13-acetate (PMA) increases surface immunolabeling and surface expression of 5-HT3A receptors in both Xenopus oocytes and in N1E-115 cells (55). The increase in surface expression of receptors developed rapidly, within minutes of PMA application, and preapplication of phalloidin, which stabilizes the actin polymerization, signiﬁcantly inhibited PMA potentiation of 5-HT-activated responses. This suggests that PKC modulation of 5-HT3A receptor function occurs via an F-actin-dependent mechanism, a conclusion that is consistent with observations that 5-HT3A receptors colocalize with F-actin-rich membrane domains, such as lamellipodia and microspikes of heterologous cells transiently expressing 5-HT3A receptors (57–60). A recent study has identiﬁed a speciﬁc interaction between 5-HT3A receptors and the light chain of microtubule-associated protein 1B (MAP1B-LC1) (61). The interaction with MAP1B-LC1 appears to be speciﬁc for 5-HT3A receptor subunits since no such interaction was found to occur with 5-HT3B receptor subunits. Overexpression of MAP1B-LC1 reduced expression levels of 5-HT3A receptors at the cell surface. Disruption of the interaction between MAP1B-LC1 and 5-HT3A receptors by a speciﬁc LC1 antisense oligonucleotide resulted in a drastic modiﬁcation of 5-HT3A channel kinetics (62). Given that neurotransmitter release can be regulated through an actin-dependent mechanism in the CNS (57), and that 5-HT3A receptors can modulate the release of dopamine and GABA in some important brain areas, it seems possible that enhancement of 5-HT3 receptor function and trafﬁcking by PKC activation may play an important role in modulating the efﬁcacy of serotonergic synaptic transmission, the release of neurotransmitters and other 5-HT3 receptor-mediated phenomena.

ALLOSTERIC MODULATORS There are a number of substances that are able to modulate 5-HT3 receptor responses, including alcohols, anesthetics, cannabinoids, steroids, and divalent cations, as well as a range of structurally diverse compounds such as ifenprodil and 5-hydroxyindole (2). The mechanism of action of most of these compounds is not yet understood, as described in more detail below. Alcohol A large body of evidence has suggested that 5-HT3 receptors are a major target for alcohol action in the CNS (9,63,64). Ethanol and short-chain alcohols can potentiate the function of cloned and native 5-HT3 receptors within

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an intoxicating concentration range (65–67), indicating that potentiation of 5-HT3 receptors could contribute to acute intoxication. The potentiation by ethanol appears to be speciﬁc for 5-HT3A receptor subunits; the efﬁcacy of such potentiation substantially decreases with inclusion of 5-HT3B receptor subunits (68,69). The precise molecular mechanisms underlying alcohol modulation of 5-HT3 receptor function are not totally understood. The potentiation is dependent on agonist concentrations: with increasing agonist concentrations, the magnitude of potentiation decreases (65,70). There is strong evidence to indicate that alcohols and anesthetics act via channel gating other than agonist binding. First, both ethanol and anesthetics enhance the current activated by receptor-saturating concentrations of dopamine, a weak partial agonist of 5-HT3 receptor (71,38). Second, kinetic analysis using electrophysiological recording has demonstrated that alcohols increase the rate of channel activation, while decreasing the rates of channel desensitization and deactivation. This observation suggests that alcohols can prolong the time constant of channel closure by stabilizing the opening state of the channel (72). This conclusion is supported by a molecular study, which identiﬁed a pre-transmembrane segment 1 (TM1) residue as an important site for the coupling process between agonist binding and gating (73). Point mutations of R222 produced constitutively active channels, which were sensitive to ethanol in the absence of agonist (70). These observations favor the hypothesis that alcohols act via channel gating independent of ligand occupancy. In contrast to the potentiation of 5-HT3A receptors, ethanol and volatile anesthetics inhibit nACha7 receptor–mediated responses (74,75). Taking advantage of the differential sensitivity of these receptors to alcohol and anesthetics, a chimeric receptor constructed from the N-terminal domain of the nACha7 receptor subunit and transmembrane and carboxyl domains of the 5-HT3A receptor subunit was used to determine the N-terminal domain that mediates sensitivity to ethanol and volatile anesthetics (74,75). A subsequent study has suggested that the M2 domain is also involved in alcohol alterations of receptor function (64,76). Point mutations of residue 294 in this domain greatly reduced the ethanol and trichloroethanol–induced potentiating effects in both Xenopus ooyctes and HEK293 cell-expression systems (64,76). Anesthetics Volatile anesthetics, such as halothane and isoﬂurane, potentiate 5-HT3 receptor-mediated currents at subclinical concentrations, and there is also evidence of modulation by a range of other anesthetics including etomidate, ketamine, and methohexital [see (77) for review]. Studies using the chimeric a7-nACh-5-HT3 receptor indicate that they may act at the extracellular Nterminal domain (75). As for alcohol effects, the potentiation by anesthetics appears speciﬁc for 5-HT3A receptor subunits, as it is less in the presence of 5-HT3B receptor subunits (68,69). Local anesthetics may also mediate some

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of their effects through 5-HT3 receptors: procaine, tetracaine, bupivacaine, lidocaine, and QX222 have been reported to inhibit 5-HT3 receptor function (see Ref. 2 for review). Cannabinoids The effects of cannabinoids, such as tetrahydrocannabinol (THC) and anandamide, are mediated primarily through cannabinoid receptors, but both of these compounds also inhibit 5-HT3 receptor function. This inhibition has been reported in neurons and cell lines expressing 5-HT3A receptor subunits, where it appears to be CB1 receptor-independent (78–80). The EC50 of THC inhibition of human 5-HT3A receptors is 38 nM, a concentration well below the human plasma concentration after smoking small amounts of cannabis (81). THC and anandamide did not alter receptor binding afﬁnity, indicating that their effects are probably mediated via channel gating (80). The direct inhibition of 5-HT3 receptors by THC may contribute to clinical implications of this compound such as the control of pain and emesis. Steroids A number of steroid hormones that interact with Cys-loop receptors have been identiﬁed (so-called neuroactive steroids) and some of these also appear to modulate 5-HT3 receptor function. Thus, the gonadal steroids 17-bestradiol and progesterone, for example, appear to act as noncompetitive antagonists, with IC50 values similar to those observed for their inhibitory action on glycine and nACh receptors, and inhibition with 17a-estradiol, mestranol, testosterone, and allopregnanolone, although not pregnanolone, has been reported (82). There is some evidence to support the proposal that steroids insert into the plasma membrane and modulate the receptor at the receptor–membrane interface in a structure-speciﬁc manner, but there are currently no molecular details on how this might occur. It is also not clear if the effect on 5-HT3 receptors is physiologically relevant—the concentrations at which these compounds are effective exceed normal physiologically relevant levels, but it is possible that ﬂuctuation in gonadal steroids may play a role in some behavioral disorders and nausea, for example, during pregnancy. Cations Cations permeate 5-HT3 receptor channels, but in addition they modulate receptor function. Thus, Ca2þ acts as an inhibitor, and probably acts both within the pore and at the binding site (83,84). A Ca2þ binding site close to the neurotransmitter binding site has been identiﬁed in AChBP, and is thought to lie in a similar location on the nACh receptor (85). The strong similarity between the structures of these proteins and the 5-HT3 receptor

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extracellular domain suggests that an N-terminal Ca2þ binding site, if it exists, will be in much the same location in the 5-HT3 receptor, although there is currently no experimental evidence to support this hypothesis. Ca2þ may also modulate 5-HT3 receptor responses via Ca2þ binding proteins such as calcineurin, which has been shown to potentiate receptor desensitization (86). Mg2þ and other divalent cations, including Cd2þ, Cu2þ, and Zn2þ, also modulate 5-HT3 receptor responses (84,87–89). Like Ca2þ, these may act at multiple sites, although the effects of Zn2þ appear more complex, with both inhibition and enhancement being reported (88,89). Other A range of other compounds also may modulate 5-HT3 receptor activity. The L-type Ca2þ channel antagonists verapamil, diltiazem, and nimodipine all inhibit 5-HT3 receptor function (90), and a more detailed study on the action of diltiazem shows that it acts as a channel blocker (91). Other compounds that inhibit 5-HT3 receptors include tetraethylammonium, r-conotoxin GVIIIA, phenothiazines, ifenprodil, bisindolylmaleimide, and 5-hydroxyindole, although this latter compound only inhibits at high concentrations and it enhances the response by reducing the rate of 5-HT3 receptor desensitization at lower concentrations (see Ref. 2 for review). THERAPEUTIC POTENTIAL The main therapeutic targets of 5-HT3 receptor active agents are currently IBS, emesis resulting from chemotherapy or radiotherapy, and in the treatment of multiple sclerosis (92–94). However, there are potentially other disorders where 5-HT3 receptor antagonists and/or agonists could be useful. Some of these are discussed below. Antiemesis The most important clinical application of 5-HT3 receptor antagonists is in the treatment of emesis and nausea, two of the most distressing side effects of chemotherapy and radiotherapy (92,93). 5-HT3 receptor antagonists have also been used for the treatment of postoperative nausea and vomiting induced by the use of halogenated volatile anesthetics (95). In clinical studies, 5-HT3 receptor antagonists have exhibited an excellent efﬁcacy in the control of the acute phase of nausea and emesis induced by emetogenic chemotherapeutic agents with few adverse side effects (11). The emetogenic agents and radiation can promote the release of 5-HT from enterochromafﬁn cells within the wall of the intestine and neurons in the brainstem, and it is likely that 5-HT3 receptor antagonists exert their antiemetic effect by competitively inhibiting serotonin at its binding sites located centrally in the dorsal medulla of the brainstem and in the intestine (96–98).

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Antinociception There is growing evidence that both central and peripheral 5-HT3 receptors are involved in the modulation of sensitivity to certain types of pain, especially inﬂammatory pain in animal models. While activation of 5-HT3 receptors by peripheral injection of 5-HT can evoke acute pain (99), central serotonergic circus contributes to the nociceptive process and pain by facilitating the activity of spinal 5-HT3 receptors (10). Nevertheless, animal studies suggest that selective 5-HT3 receptor antagonists are effective for the treatment of inﬂammatory stimuli and altered pain perception in chronic pain (100). However, whether or not 5-HT3 receptor antagonists can be used as antinociceptive agents in humans remains to be determined. IBS and the Motility of the Gastrointestinal Tract IBS is the most comment chronic disorder of the gastrointestinal function, affecting a signiﬁcant minority of the general population. The disease is characterized by continuous or remittent abdominal pain and is associated with altered bowel habits, diarrhea or constipation, or both (101,102). The precise pathophysiology of IBS remains elusive, perhaps involving neurotransmission within the brain–gut axis (102,103). It is well established that the antagonism of 5-HT3 receptors is a valuable therapeutic approach for the treatment of IBS, especially for female patients (104). Alcohol and Drug Abuse and Withdrawal Local activation of 5-HT3 receptors can increase somatodendritic dopamine release in the ventral tagmental area and nucleus accumbens. Administration of 5-HT3 receptor antagonists signiﬁcantly attenuates the effects of alcohol and other drugs of abuse. Ondansetron, for example, a highly selective and potent 5-HT3 receptor antagonist, has been shown to reduce alcohol consumption in animal studies (9,105) and to be an effective treatment for early onset alcoholism (106,107). Ondansetron has also been found to reduce morphine self-administration in both native and morphine dependent rats. Micro-injection of ondansetron into the rat nucleus accumbens reduced the stimulatory effect of cocaine-induced locomotor activity (108).

CONCLUSION The 5-HT3 receptor is a typical Cys-loop receptor in that it can be allosterically affected by a range of modulators. For many of these, little is known about the mechanism of action, but given the potential for therapeutic intervention, we anticipate much research in this area in the future.

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84. Niemeyer MI, Lummis SC. The role of the agonist binding site in Ca(2þ) inhibition of the recombinant 5-HT(3A) receptor. Eur J Pharmacol 2001; 428: 153–161. 85. Celie PH, van Rossum-Fikkert SE, van Dijk WJ, Brejc K, Smit AB, Sixma TK. Nicotine and carbamylcholine binding to nicotinic acetylcholine receptors as studied in AChBP crystal structures. Neuron 2004; 41:907–914. 86. Yakel JL. Calcineurin regulation of synaptic function: from ion channels to transmitter release and gene transcription. Trends Pharmacol Sci 1997; 18: 124–134. 87. Lovinger DM. Inhibition of 5-HT3 receptor-mediated ion current by divalent metal cations in NCB-20 neuroblastoma cells. J Neurophysiol 1991; 66: 1329–1337. 88. Gill CH, Peters JA, Lambert JJ. An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells. Br J Pharmacol 1995; 114:1211–1221. 89. Hubbard PC, Lummis SCR. Zn(2þ) enhancement of the recombinant 5-HT(3) receptor is modulated by divalent cations. Eur J Pharmacol 2000; 394:189–197. 90. Hargreaves AC, Gunthorpe MJ, Taylor CW, Lummis SC. Direct inhibition of 5-hydroxytryptamine3 receptors by antagonists of L-type Ca2þ channels. Mol Pharmacoll 1996; 50:1284–1294. 91. Gunthorpe MJ, Lummis SC. Diltiazem causes open channel block of recombinant 5-HT3 receptors. J Physiol 1999; 519:713–722. 92. Gaddum JH, Hameed KA. Drugs which antagonize 5-hydroxytryptamine. Br J Pharmacol Chemother 1954; 9:240–248. 93. Humphrey PP, Bountra C, Clayton N, Kozlowski K. Review article: the therapeutic potential of 5-HT3 receptor antagonists in the treatment of irritable bowel syndrome. Aliment Pharmacol Ther 1999; 13(suppl 2):31–38. 94. Macleod AD. Ondansetron in multiple sclerosis. J Pain Symptom Manag 2000; 20:388–391. 95. Apfel CC, Kranke P, Katz MH, et al. Volatile anaesthetics may be the main cause of early but not delayed postoperative vomiting: a randomized controlled trial of factorial design. Br J Anaesth 2002; 88:659–668. 96. Andrews PL, Rapeport WG, Sanger GJ. Neuropharmacology of emesis induced by anti-cancer therapy. Trends Pharmacol Sci 1988; 9:334–341. 97. Cubeddu LX, Hoffmann IS, Fuenmayor NT, Finn AL. Efﬁcacy of ondansetron (GR 38032F) and the role of serotonin in cisplatin-induced nausea and vomiting. N Engl J Med 1990; 322:810–816. 98. Schworer H, Racke K, Kilbinger H. Cisplatin increases the release of 5-hydroxytryptamine (5-HT) from the isolated vascularly perfused small intestine of the guinea-pig: involvement of 5-HT3 receptors. Naunyn Schmiedebergs Arch Pharmacol 1991; 344:143–149. 99. Richardson BP, Engel G, Donatsch P, Stadler PA. Identiﬁcation of serotonin M-receptor subtypes and their speciﬁc blockade by a new class of drugs. Nature 1985; 316:126–131. 100. Farber L, Haus U, Spath M, Drechsler S. Physiology and pathophysiology of the 5-HT3 receptor. Scand J Rheumatol Suppl 2004; 119:2–8.

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7 Nicotinic Receptors R. C. Hogg and D. Bertrand Department of Neuroscience, Centre Me´dical Universitaire, Geneva, Switzerland

THE nAChR AS A PROTOTYPE OF AN ALLOSTERIC PROTEIN The allosteric model was ﬁrst proposed by Monod Wyman and Changeux in 1965 to explain the observed behavior of proteins such as hemoglobin (1). This model was applied to ligand-gated ion channels by Karlin in 1967, who proposed that it could be extended to describe the functioning of the acetylcholine (ACh) receptor (2). The ubiquitous nature of the nicotinic acetylcholine receptor (nAChR) at the neuromuscular endplate, the discovery of the selective ligand a-bungarotoxin, and the high density of nAChRs found in the electric organ of the eel Electrophorus electricus resulted in these receptors being extensively studied and becoming a model for the functioning of ligand-gated ion channels. Numerous kinetic schemes have been proposed to describe their behavior; structurally, the nAChR conforms to the requirements of the model proposed by Changeux et al. (3) comprising ﬁve subunits arranged around a central ion-conducting pore, which confer axes of pseudosymmetry. This model can describe the behavior of a protein made up of a number of subunits, which contain at least two equivalent binding sites and can exist in multiple conformational states. Ligand binding can occur to any of the states and, when present, the ligand binds preferentially to the desensitized and active open states. The nAChR family is composed of several receptor subtypes, which are differentially expressed throughout the organism. Different combinations of subunits give rise to receptors with distinct pharmacological and

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biophysical properties. In Torpedo electric organ and vertebrate fetal muscle, the subunit composition is (a1)2b1cd, and in adult muscle the c subunit is replaced by an e subunit (4). The order of subunits is highly constrained with a clockwise sequence of a1ea1b1d. In addition to the receptors found at the neuromuscular junction, there is also a family of nAChRs expressed in the nervous system. To date, 12 neuronal nAChR subunits have been described, a2 to a10 and b2 to b4. The assembly of subunits in the neuronal nAChRs is less tightly constrained than that of the muscle receptor. Moreover, a7, a8, and a9 subunits can form functional homomeric pentamers when expressed in mammalian or amphibian cells; however, a9 preferentially coassemble with a10 (5). The other subunits combine in a putative (a)2(b)3 stoichiometry to form functional channels (6), with the exception of b3 and a5, which do not form functional receptors when expressed alone or with a single type of a or b subunit. The majority of receptor complexes identiﬁed contained one type of a and one type of b subunit; however, heteromeric ‘‘triplet’’ receptors involving three types of subunits can form in the Xenopus oocyte expression system (7–9). Also, the precise subunit composition of b3- and a5-containing receptors in vivo has yet to be conﬁrmed. Studies involving site-directed mutagenesis of the nAChR have identiﬁed the region of the receptor and the amino acid residues involved in binding ACh (10–12). Recently, the crystal structure of a homomeric ACh-binding protein, which is homologous to the extracellular domain of the nAChR, has been published, providing a three-dimensional view of the ACh binding site at the atomic resolution (13). The strongest interactions with ACh are with residues located on the a subunit that form the ‘‘principal’’ component of the binding site; weaker interactions with residues on the adjacent b, in the case of neuronal receptors, or the neighboring e and d subunits in the muscle receptor, form the ‘‘complementary’’ binding site. Thus, the muscle receptor and heteromeric neuronal nAChRs with a (a)2(b)3 subunit stoichiometry are supposed to have at least two ACh binding sites. In homomeric receptors, the complementary component is contributed by the adjacent a subunit, resulting in ﬁve identical ACh binding sites; however, it is not known as to how many of these sites need to be occupied to stabilize the receptor in the open conformation. The allosteric model predicts that the protein can exist in multiple states and undergoes spontaneous conformational transitions, involving ‘‘rigid body movement’’ of the subunits, between a minimum of two or more preexisting conformational states (14). These different states each display a different afﬁnity for a ligand. A prediction of this model is that transitions from one state to another depend upon both the presence of a ligand and/or the isomerization coefﬁcient (L0–L2), which is representative of the free energy change for the transition. Spontaneous transitions between states in the absence of agonist have been conﬁrmed both at the muscle nAChR (15) and at a7 mutant receptors (16).

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For the nAChR, a minimum of three states must be included in the model to describe its function (Fig. 1A). At rest, in the absence of agonist, the equilibrium among these conformational states is in favor of the resting (R) closed state; Exposure to low agonist concentrations stabilize the receptor in the D state; this can be observed following preincubation with low agonist concentrations, which do not lead to receptor activation (Fig. 1B), indicating that the isomerization coefﬁcient L1 must be lower than L0. Exposure to higher agonist concentrations preferentially stabilizes the receptor, ﬁrst in the active (A) open state. In the continued presence of agonist, the

Figure 1 The minimal three-state allosteric model that can account for the functional properties of the nAChR. (A) The resting, open and desensitized conformations of the receptor are represented, transitions between states are determined by the isomerization constants L0–L1. (B) In the absence of agonist, the channel remains closed in the resting state; when a high concentration of agonist is applied, the receptor is stabilized brieﬂy in the active conformation and in the continued presence of agonist the receptor is progressively stabilized in the desensitized state. (C) The desensitized closed state of the receptor presents a higher afﬁnity for ACh than the active state, low agonist concentrations stabilize the receptor in the desensitized state without activating it. (D) The concentration dependence of this effect in the continued presence of agonist is shown by the dashed line. The solid line represents the concentration dependence of receptor activation by brief exposure to agonist when not in the continued presence of agonist. Abbreviation: nAChR, nicotinic acetylcholine receptor.

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Figure 2 (Caption on facing page)

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receptors are progressively stabilized in the D state as seen in Figure 1C. In the D state, the receptor is closed, despite the bound agonist, a condition that is somewhat analogous to the inactivated state of voltage-gated channels. While desensitization is thought to play little role in synaptic transmission at the neuromuscular junction, it is likely to be important in the central nervous system (CNS) with, for example, modulatory effects involving extrasynaptic nAChRs. RECEPTOR MODULATION BY ALLOSTERIC LIGANDS Allosteric modulators are molecules that modify receptor properties through interaction at sites on the receptor that are distinct from the binding site for the natural agonist. The term ‘‘allosteric binding site’’ refers to a site of interaction, which is structurally distinct from the natural agonist binding site or ‘‘orthosteric’’ site. An allosteric effector binding at such a site can change the agonist efﬁcacy, and/or binding afﬁnity, at the orthosteric site and have either a positive or a negative effect on receptor function (1,17). Binding of a molecule to an allosteric site alters the isomerization coefﬁcient (L) and therefore the equilibrium between conformational states. Ligands that displace the equilibrium in favor of the active (open) state by reducing the isomerization coefﬁcient L0 are termed positive allosteric effectors (Figs. 1A and 2A). While these compounds are not agonists and do not activate the receptor when applied alone, they potentiate the responses to an agonist by increasing the probability of channel opening. Similarly, compounds that displace the equilibrium in favor of the closed state by increasing L0 are termed negative allosteric effectors. As the effects of allosteric modulation are not restricted to any particular state or isomerization coefﬁcient, multiple outcomes can be predicted as a function of the isomerization coefﬁcients that are affected by the modulator.

Figure 2 (Figure on facing page) Point mutations and allosteric effectors can change receptor properties by altering the isomerization constants L0–L2. (A) The effect of altering the L0 on the agonist concentration–response curve, lowering of L0 causes a shift of the curve to the left, increase the slope, and increases the Emax, whereas increasing L0 causes a rightward shift in the curve, reduction of the slope, and a decrease in Emax. Another prediction of the model is that at low values of L0 spontaneous channel opening will occur. (B) The effects of the L247T mutation can be accounted for by assuming that the desensitized state of the receptor is conducting; low concentrations of agonist, which would normally cause receptor desensitization without activation, cause activation of the L247T receptor. (C) Typical ACh-evoked currents recorded in cells expressing either the control (left) and L247T (right) a7 receptors (for clarity current amplitude have been normalized to unity). Note the differences in response proﬁle and absence of desensitization of the L247T mutant.

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THE a7 MODEL The discovery of homomeric nAChRs opened new possibilities for sitedirected mutagenesis aimed at understanding the relationships between receptor structure and function. The main advantage of homomeric receptors is that a single point mutation will be present in all ﬁve subunits that compose the functional receptor and therefore reduces the number of permutations and constructs to be realized. Mutations in the pore region of the a7 nAChR have been shown to have signiﬁcant effects on receptor properties (18,19). In a ﬁrst series of experiments, mutations in the second transmembrane domain (TM2) revealed surprising effects that could be best interpreted using an allosteric model. Among these mutants it is worth examining the effects caused by mutation of the valine at position 251 in the chick a7 receptor. Shortening of the lateral chain of the amino acid at position 251 in the mutant V251T was characterized by an increased sensitivity to ACh and reduced receptor desensitization. The effects of this mutation could be interpreted as causing a lowering of the energy barrier (L0), which governs transitions between the resting and active states (18,19). Such a hypothesis can account for both the change in agonist efﬁcacy and altered desensitization properties, as these are dictated by the equilibrium constants (L0–L2). Mutation of the canonical leucine found at position 247 in the chick a7 receptor introduces pleiotropic effects (20). Substitution of leucine by threonine yielded the mutant L247T that is characterized by a 200-fold increase in sensitivity to ACh, loss of desensitization, loss of rectiﬁcation, increase of single channel conductance, and conversion of competitive antagonists into full agonists (18–20). Whereas it is not expected that a point mutation can affect properties of the receptor that are so diverse, all these effects can be accounted for using the model proposed in Figure 2B. That is, it is sufﬁcient to assume that the L247T mutation converts the properties of the desensitized (normally closed) state into a functional open state. On the basis of this hypothesis, exposure to any compound that stabilizes the desensitized state will result in the activation of the mutant receptor and can now be monitored as an opening of the receptor. This explains why competitive antagonists, which bind to the desensitized state of the receptor, behave as agonists at receptors containing the L247T mutation. It is well documented that there are discrepancies of several orders of magnitude between agonist binding afﬁnities and the apparent afﬁnities observed in physiological experiments, with agonist binding having a higher afﬁnity than functional effects. This difference is generally explained by the fact that binding experiments are carried out over long exposure times (minutes to hours), whereas physiological responses are measured over milliseconds to seconds, thus the conformation of the receptor differs between the two conditions. Functional experiments have shown that prolonged

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exposure to low agonist concentrations can displace the receptor equilibrium toward a desensitized state without detectable activation of the receptors. This indicates that afﬁnity of the ligand for the desensitized state must be higher than for the active state. It has been demonstrated that the desensitized conformation of the nAChR presents a higher-afﬁnity binding site for ACh than the resting state (21,22). Thus, in binding experiments it is assumed that the ligands progressively stabilize the receptor in the desensitized state and therefore reﬂect the afﬁnity of this particular state. In functional studies, physiological responses can only be observed when relatively high concentrations of agonists are applied for a brief time, conﬁrming that the afﬁnity of the active (open) state is lower than that of the desensitized state. Another consequence of this model is that compounds that stabilize the resting state should remain antagonists at the L247T mutant whereas compounds that stabilize the D state, and which are otherwise antagonist at the wild-type receptor, should activate this mutant. Consequently, if we assume that the desensitized state of the mutant L247T receptor is conducting, the receptor would display a higher sensitivity to agonists, and that the concentration dependence of activation should reﬂect the afﬁnity of the desensitized state. Changes in rectiﬁcation and conductance properties of the receptor can be accounted for by the modiﬁcation of L247 amino acid that is facing the lumen of the ionic pore. It is interesting to note that multiple effects caused by a point mutation can be accounted for by a single hypothesis when considering the a7 nAChR as a prototype of allosteric protein. Moreover, a prediction of this model is that a lowering of the isomerization coefﬁcient between the resting closed and desensitized open state by the L247T mutation will give rise to spontaneous opening of the receptor. Thus, a signiﬁcant fraction of the receptors should open in the absence of agonist. In agreement with this prediction, spontaneous activity of the L247T mutant was reported (16). While it is beyond the scope of this work to examine the effects of each and every mutation studied in the nAChR, it is important to bear in mind that any modiﬁcation of the equilibrium constants will not only affect agonist and antagonist sensitivity, but also result in alteration of the modulation by allosteric effectors. INFLUENCE OF RECEPTOR SUBUNIT COMPOSITION ON RECEPTOR PROPERTIES The location of the binding site for ACh at the interface between two adjacent subunits implies that in the muscle receptor the binding sites are nonequivalent with the ad interface having a higher afﬁnity than the ac (23). It has been demonstrated that it is necessary for two molecules of agonist to be bound to the receptor to stabilize the open active conformation of this receptor, and that there is a cooperativity of binding, with the afﬁnity for the second molecule of ACh increasing after the ﬁrst has bound (24,25).

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Because the ACh binding site is formed by both the a and its adjacent subunit, the subunit composition of a receptor determines its pharmacological properties; thus, it is important to compare the activity of allosteric modulators on different nAChR subunit combinations. Differing effects at different receptor subtypes can indicate structural features, which may be important for modulator activity. The inﬂuence of the subunit composition of the receptor on the modulatory effects of an allosteric ligand are illustrated in the following sections, which discuss the effects of modulators such as the steroid 17b-estradiol and Zn2þ on heteromeric nAChRs.

ALLOSTERIC MODULATORS Modulation of nAChRs by Steroids A number of natural steroids, which are locally synthesized in the brain, are known to interact with ligand-gated channels and may represent an important physiological mechanism by which receptor function is regulated in vivo. It was originally thought that as steroids are highly lipophilic they might modulate ion channel activity by disruption of the lipid membrane environment. However, many steroids have been demonstrated to interact directly with ion channel proteins. Promegestone is a noncompetitive antagonist at the Torpedo nAChR (26), and progesterone inhibits neuronal nAChRs from avian brain expressed in Xenopus oocytes in a noncompetitive manner (27,28). The inhibitory activity of steroids was inversely related to their lipophilicity, indicating their effects are not caused solely by a perturbation of the lipid membrane, but are likely to involve an interaction with the receptor protein (29,30). In addition, progesterone coupled to bovine serum albumin (BSA), which is water soluble and unable to partition into the plasma membrane, was effective at inhibiting ACh-evoked currents at a4b2 receptors (28). Steroids did not affect binding of radiolabeled cytisine to cell homogenates containing neuronal a4b2 receptors, and steroid effects at the nAChR were not affected by the presence of agonists (31), indicating an allosteric site of interaction. The relationship between the structure and activity of various steroids at nAChRs and the selectivity of some steroids between receptor subtypes has helped to clarify some of the molecular interactions involved. The inhibitory effect of 3a,5a,17b-3-hydroxyandrostane-17-carbonitrile was enantioselective, indicating the site of interaction on the nAChR is stereoselective and potency was found to depend strongly on the orientation of the group at position 17. 17b-Estradiol potentiated ACh-activated currents through human neuronal a4b2 and a4b4 receptors expressed in Xenopus oocytes by increasing the apparent afﬁnity of the receptor for ACh (32,33). This effect was rapid in onset, concentration-dependent, and readily reversible, further that the site of action on the protein is external. 17b-Estradiol also increased

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the probability of opening of human a4b2 nAChRs expressed in HEK 293 cells. The effect of 17b-estradiol showed enantioselectivity; an unsaturated A ring and a free hydroxyl group at positions 3 and 17 were necessary for activity (32), again suggesting that effects are not mediated by a disruption of the membrane lipid environment. 17b-Estradiol had no effect at a3b2 or a3b4 receptors, illustrating that the a4 subunit was necessary for these effects. It has been reported that human but not rat a4b2 receptors were potentiated by estradiol (32). This species and subunit selectivity has enabled the identiﬁcation of the region of the receptor protein required for modulation. Rat and human a4 subunits differ in the C-terminal tail of the a4 subunit, pointing to a possible location of the steroid binding site. Chimeric subunits containing the N-terminal domain of the a4 subunit and the C-terminal of the a3 subunit were not potentiated by 17b-estradiol, whereas the a4 C-terminal attached to an a3 N-terminal was potentiated by 17b-estradiol. Moreover, truncation of the C-terminal tail of the a4 subunit abolished the effects of 17b-estradiol (33). Mutation of individual residues in the C-terminal as well as extending the terminal sequence by inserting residues inhibited potentiation by estradiol, indicating that the nature and position of the ﬁnal four residues were critical (32). The putative 17b-estradiol binding site on the C-terminal was found to be distinct from the progesterone binding site (32), suggesting that individual steroids interact with different sites on the receptor protein. It should be mentioned at this point that mutations in the receptor, which affect the activity of an allosteric modulator, do not necessarily indicate the site of interaction between the modulator and receptor. Although single amino acid mutations in the pore lining M2 domain, such as the L247T and V251T mutations in the chick a7 receptor, change the sensitivity of the receptor to the natural ligand ACh, this does not necessarily reﬂect an alteration of the afﬁnity of the receptor for ACh. A distinction should be maintained between binding afﬁnity, measured from binding studies, which reﬂects the afﬁnity of binding to the receptor, and apparent afﬁnity from functional studies, which could represent an increase in the efﬁcacy of ACh, due to an alteration of L0. Mutations in the M2 domain have been demonstrated to change the sensitivity of the receptor to allosteric modulators. These mutations alter the energy barrier for transitions between states and thus the proportion of receptors that exist in a particular state at equilibrium. And since allosteric modulators bind with high afﬁnity to certain states of the receptor, it can be predicted from the model in Figure 1A that the sensitivity to allosteric modulators will be altered by these mutations. Modulation by Zinc Modulation of the nAChR by low concentrations of Zn2þ is also dependent on the subunit composition of the receptor. a2b2, a2b4, a3b4, a4b2, and

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a4b4 receptors were potentiated by low concentrations of Zn2þ, whereas a3b2 receptors were inhibited (34,35). Zn2þ also inhibited homomeric a7 receptors (36), suggesting that a property of the a2 and a4 subunits, which is not present on the a3 and a7 subunits, conveys the Zn2þ-dependent modulation. The effects of Zn2þ were biphasic, with concentrations of Zn2þ greater than 100 mM inhibiting currents. The effects of Zn2þ were attenuated by diethylpyrocarbinate or by lowering the pH to 5.5, suggesting that Zn2þ may be interacting with histidine residues. In an attempt to identify the residues involved, Hsaio et al. used chimeric a3–a4 subunits coexpressed with the b4 subunits. Both a3–216–a4, comprising the N-terminal domain of a3 joined to the transmembrane domains of a4, and the opposite a4–216–a3 chimeras were less sensitive to potentiation by Zn2þ; however, loss of sensitivity in the a3–216–a4 chimera was much greater. This was taken to be an indication that most of the critical determinants for Zn2þ potentiation are located in the N-terminal region of the a4 subunit, but Zn2þ must also have additional sites of interaction on the protein. However, caution must be exercised in the interpretation of these results; as mentioned previously, mutations of the receptor can have multiple effects on receptor behavior, including sensitivity to allosteric modulators, and therefore does not necessarily indicate the site of interaction. Zn2þ is stored in presynaptic vesicles and is released into the synaptic cleft. High concentrations of Zn2þ-containing neurons are found in the cerebral cortex and limbic system (37), and the concentration of Zn2þ in the synaptic cleft during neurotransmission has been estimated to rise as high as 300 mM (38). Due to the biphasic concentration-dependent effect, Zn2þ can both enhance and reduce nAChR function, depending on the frequency of release of Zn2þ from the presynaptic neuron. In addition, Zn2þ has also been reported to modulate other ligand-gated ion channels (39–42) and may represent an important endogenous allosteric modulator.

POSITIVE ALLOSTERIC EFFECTORS Acetylcholinesterase Inhibitors A number of cholinesterase inhibitors such as physostigmine, galanthamine, and tacrine can modulate nAChRs via an allosteric mechanism. These compounds potentiate ACh-evoked responses at low concentrations and cause open channel block at higher concentrations, resulting in a bell-shaped dose–response curve (43–45). Tritiated physostigmine was shown to label residue Lysine-125 on the a subunit of the Torpedo receptor, indicating that its site of interaction is distinct from the ACh-binding region (46). Tacrine and physostigmine have similar potentiating effects on neuronal a4b2 and a4b4 receptors; however, at these receptors, effects were competitive with ACh and with 125I-epibatidine, suggesting that at low concentrations of

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modulator and agonist, tacrine and physostigmine may interact with one of the two ACh binding sites, while the other is occupied by ACh, and cause a synergistic potentiation of the response (44). When the concentration of ACh is increased, the potentiating effect is lost as ACh displaces the modulator. Several studies have also demonstrated a potentiating effect of the acetylcholinesterase inhibitor galanthamine, via an allosteric mode of action. Galanthamine, like tacrine and physostigmine, acts as an open channel blocker at high concentrations, giving a bell-shaped dose–response curve, and shifts the dose–response curve for ACh to the left, without changing the amplitude of the maximal response. This suggests that the afﬁnity for ACh may be increased without affecting the intrinsic properties of the receptor. Galanthamine has been demonstrated to have beneﬁcial effects in the treatment of Alzheimer’s disease, which may be attributed to a restoration of cholinergic function; however, it is not known if this is due to the anticholinesterase, allosteric modulator properties of the molecule. It should be kept in mind that several weeks of galanthamine treatment are required before any beneﬁcial effects are observed; however, the cause of this delay in effect has not been elucidated. Anthelmintics It has been proposed that the anthelmintic action of ivermectin is due to the activation of glutamate channels in nematodes, which causes a reduced excitability of the muscle cells (47); however, ivermectin was also found to be an allosteric potentiator of human and chick a7 receptors expressed in Xenopus oocytes and K-28 cells (48). In agreement with the effects predicted for a positive allosteric modulator, ivermectin shifted the ACh concentration–response curve to the left and increased the slope of the curve. The amplitude of the response to a maximal concentration of ACh was increased approximately threefold. Investigation of the effects of ivermectin on a7 receptors with the V251T and L247T mutations has provided some insight into the mechanism of ivermectin potentiation of ACh-evoked responses. In a7 receptors containing the V251T mutation, ivermectin shifted the concentration– response curve for ACh to the left, without increasing the maximal amplitude of the current evoked by a maximal concentration of ACh. In contrast, at L247T mutant receptors, ivermectin had no effect on the EC50 value, but caused a reduction in amplitude of the responses to ACh. Using the three state allosteric model, these effects of ivermectin on mutated receptors can be explained if we assume that the L247T mutation renders a desensitized state conducting, whereas the V251T mutation alters the equilibrium between the R and A states by affecting the allosteric constant (L0) for the transition. Thus, if ivermectin lowers L0 for the transition from R to A in the a7 L247T, we should see a shift in the equilibrium of the pool of receptors, from the high-afﬁnity desensitized open state, to the lower afﬁnity A state and a loss

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of the potentiating effect at this mutant. However, at receptors with the V251T mutation, ivermectin can further lower L0, facilitating transition to the A state and increasing the afﬁnity of the receptor for ACh without affecting the transition to the desensitized state, and thus having no effect on the maximal current. Another anthelmintic, levamisole, exerts its effects by acting as an agonist at nAChRs on the soma of nematode muscle cells and causing a spastic paralysis (47). Levamisole is also a potentiator of agonist-induced responses at human a3b2 and a3b4 receptors. Competition experiments with ACh indicate that the site of action is not competitive, indicating an allosteric mode of action; however, there is presently no information on the region of the receptor that is determinant for the activity of levamisole. 5-Hydroxyindole 5-Hydroxyindole (5-HI) is another molecule that potentiates currents at recombinant human a7 receptors (49). 5-HI also increased ACh-induced calcium signals in Fura-2 loaded cells (rat pituitary adenoma GH4) expressing a7 receptor cells and at native a7 receptor human neuroblastoma IMR-32 cells. ACh-induced glutamate release in rat cerebellar slices was also potentiated by 5-HI, indicating that 5-HI has similar effects on native receptors. The potentiating effects of 5-HI were reduced by selective a7 antagonists. 5-HI has also been reported to potentiate currents at 5-HT3 receptors. Mutation of the 60 threonine in the pore of the a7 receptor to a phenylalanine, T244F, caused a loss of sensitivity to 5-HI (50). T244F mutant receptors showed increased sensitivity to ACh, an increase in the amplitude of the maximal ACh-evoked current, and slowed desensitization. Serum Albumin Conroy et al. (51) demonstrated that BSA enhances ACh-evoked currents at a7 receptors in chick ciliary ganglion and rat hippocampal neurons. BSA shifted the dose–response curve for ACh to the left with an increase in the slope and the amplitude of the maximal response. Single-channel studies revealed that the mechanism of the potentiation is due to an increase in the steady-state channel opening probability, caused by an increased frequency of channel opening and an increase in the mean channel open time. BSA had no effect on a-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) or N-methyl-D-aspartate (NMDA)-induced currents in hippocampal neurons. The concentration of albumin is 200 times lower in the cerebrospinal ﬂuid than in the circulation, making it unlikely that this represents a physiologically signiﬁcant mechanism of nAChR regulation. However, this is an additional demonstration of a high-afﬁnity site on the nAChR, which can cause a positive modulation of receptor function. BSA was able to overcome noncompetitive inhibition of a7 nAChRs by

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b-amyloid peptide, identifying this site as a potentially valid pharmacological target for drug treatment to overcome reduced nAChR function associated with the early stages of Alzheimer’s disease. Modulation of nAChRs by Ca21 The extracellular Ca2þ concentration has been demonstrated to have an effect on the amplitude of ACh-evoked responses; this may represent another physiological mechanism whereby the activity of nAChRs is augmented during periods of synaptic activity. Ca2þ is stored in presynaptic vesicles together with neurotransmitter, and is released during exocytosis (52). The extracellular Ca2þ concentration is normally in the region of 1 mM; however, concentrations in the synaptic cleft can rise to as high as 10 mM during periods of increased synaptic activity. Elevated extracellular Ca2þ potentiated ACh-induced currents through a7 receptors expressed in Xenopus oocytes (53). Neuronal a3b4, a2b2, a3b2, and a3b4 nAChRs expressed in Xenopus oocytes (54) and a4b2 nAChRs in HEK293 cells (55) also show potentiation of ACh-induced responses in raised extracellular Ca2þ. These effects have also been conﬁrmed in native tissue, with ACh-induced responses in neurons from the medial habenula potentiated by an elevated concentration of extracellular Ca2þ (56), and in tissue from mouse brain, Ca2þ potentiated the maximal nicotine-induced response in a concentrationdependent manner (57). It has also been reported that Ca2þ increases the probability of channel opening of native nAChRs in chromafﬁn cells (58). In cultured hippocampal neurons, extracellular Ca2þ modulates both the activation and the desensitization of a7 nAChRs. Increasing the extracellular Ca2þ concentration from a starting level of 0.01 mM increased the efﬁcacy of ACh at nAChRs with an EC50 of 0.1 mM; however, at higher concentrations (>1 mM) Ca2þ caused inactivation of nAChRs with an inhibitory concentration 50% (IC50) of 11 mM (59). Mutagenesis of the receptor protein has identiﬁed the site of interaction of Ca2þ with the nAChR. Chimeric a7-V201-5HT3 receptors containing the C-terminal end of the 5HT3-receptor are potentiated by Ca2þ, whereas native 5HT3 receptors are not (53), suggesting that Ca2þ interacts with the extracellular N-terminal part of the a7 subunit. Mutation of amino acids in the region 160–174 of the extracellular domain abolished the Ca2þ-dependent facilitation, suggesting this is the site of Ca2þ interaction. Ca2þ was also demonstrated to bind to a peptide comprising the residues 160–174 or the a7 receptor. Binding was inhibited by the mutation E172Q, indicating that this is indeed the site of interaction with Ca2þ and does not reﬂect a loss of sensitivity of the receptor to Ca2þ due to the allosteric effects of point mutations (60). In addition to the direct effects of Ca2þ on the receptor, increased entry of Ca2þ ions through a7 nAChRs may activate intracellular Ca2þ cascades, possibly giving rise to secondary effects via receptor phosphorylation.

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Modulation by Protein Kinases Phosphorylation is an important mechanism in the regulation of many ligand-gated ion channels; in addition to inﬂuencing receptor expression and subcellular localization, it can also alter channel properties such as desensitization and recovery from inactivation (61). In chick ciliary ganglion, a3, a4, a5, a7, b2, b3, and b4 subunits are phosphorylated by protein kinase A (PKA) and protein kinase C (PKC), and consensus sequences for PKA and PKC phosphorylation sites have been identiﬁed on the major intracellular loop between the M3 and M4 transmembrane segments of the rat, chick, and human a7 and a4 subunits and on two isoforms of the human a1 subunit (62). At each of these putative phosphorylation sites, the phosphorylated residue is a serine or a tyrosine. Due to the fact that this site is distinct from the natural agonist site, this modulation can be considered to have an allosteric mechanism of action. The phosphorylation of intracellular serine and tyrosine residues on the Torpedo nAChR was shown to alter receptor desensitization (11,63–68) and mutation of the serine at position 368 to an alanine in a4b2 receptors expressed in Xenopus oocytes prevented recovery from nicotine-induced desensitization (69). PKC-mediated phosphorylation of rat a7, a3b2, a4b2, and a4b4 nAChRs expressed in Xenopus oocytes by arachidonic acid potentiated the ACh-induced current (70). Src family kinases have been demonstrated to modulate nAChRs in chromafﬁn cells and a3b4a5 nAChRs in kidney cells. Inhibition of Src kinase caused a decrease in the current amplitude, whereas inhibition of phosphotyrosine phosphatase or the expression of a mutant Src kinase that was chronically active increased current amplitude (71). Given the ubiquitous nature of phosphorylation of membrane proteins and ion channels, this mechanism is likely to be highly relevant physiologically; the intracellular location of the allosteric binding site may limit its suitability as a therapeutic target. Modulation by Endogenous Peptides The discovery of endogenous peptides, which interact with and alter the functioning of nAChRs, has identiﬁed this endogenous peptides as a possible mechanism of nAChR modulation in vivo. The glycosylphosphatidylinositol (GPI)-anchored protein Lynx-1 is a member of the leucocyte antigen-6-urokinase-type plasminogen activator (Ly6-uPAR) protein family and has been isolated from the mammalian CNS, where it is expressed in pyramidal neurons of the cortex and Corne d0 Ammon (CA3) pyramidal neurons (72). Lynx-1 has been shown to form stable complexes with a7 and a4b2 nAChRs and to potentiate ACh-evoked currents and increase the rate of receptor desensitization in vitro (72,73). Secreted Ly6-uPAR related protein 1 (SLURP-1), which is a member of the same family of

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proteins, is a secreted peptide that is found in physiologically active concentrations in the circulation (74). SLURP-1 is highly expressed in keratinocytes, lung, and squamous epithelium, all of which in addition express a7 AChRs (75–77). Both Lynx-1 and SLURP-1 share a high degree of sequence homology with the three-ﬁngered snake and frog cytotoxins, such as the a-neurotoxins, suggesting that they may adopt a similar three-dimensional structure. SLURP-1 is a potent potentiator of ACh-evoked currents at human a7 receptors in vitro (78). Picomolar concentrations of SLURP-1 increased the maximum amplitude of ACh-evoked responses, shifting the concentration– response curve to the left and increasing the slope, consistent with an allosteric mode of action. a-neurotoxins inhibit ACh-evoked responses by competing for the agonist binding site; thus, despite the high degree of sequence homology and probable structural similarity that these endogenous proteins share with the a-neurotoxins, their effects as potentiators suggest that their site of action on the nAChR is not the same as that of the a-neurotoxins. Modulation of these receptors by a variety of endogenous ligands may represent a ‘‘ﬁne tuning’’ mechanism by which the activity of neuronal circuits can be subtly changed. NEGATIVE ALLOSTERIC MODULATION OF THE nAChR From a therapeutic point of view there are several advantages to using negative allosteric modulators over conventional competitive antagonists and channel blockers. The principal advantage over competitive inhibition is that the effects of a negative allosteric modulator are saturating. When all the allosteric sites are occupied, the effect is maximal and cannot be overcome by raised levels of neurotransmitter; thus, not only is the concentration–response curve displaced to the right, maximal current amplitude is also reduced. Negative allosteric modulators can also depress the maximal response to high agonist concentrations, while preserving agonist sensitivity at lower concentrations. Allosteric modulators that bind to the desensitized state will effectively reduce the number of receptors available for activation. Calcitonin Gene-Related Peptide Calcitonin gene-related peptide (CGRP) has been reported to modulate the neuronal nAChRs in rat chromafﬁn cells (79,80). Effects are dependent on the length of the peptide; the full-length 37 residue peptide and the seven amino acid N-terminal fragment had an inhibitory effect on nAChRs in rat chromafﬁn cells, whereas shorter N-terminal fragments, CGRP 1–4, 1– 5, and 1–6 potentiated nicotine-induced currents. The fragments causing a positive modulation shifted the nicotine dose–response curve to the left

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without increasing the maximal response of the receptor, consistent with an increase in the receptor sensitivity caused by a lowering of L0. Allosteric Modulators as Therapeutics The association between nAChRs and several neurological disorders (81,82) has focused attention on these receptors as novel therapeutic targets. In the last few years, there has been considerable interest in nAChRs as therapeutic targets, largely due to their association with a range of disorders. Diseases involving nAChRs can be divided into those involving a loss of cholinergic function, such as the neurodegenerative conditions Alzheimer’s and Parkinson’s disease, and those in which genetic mutations lead to the expression of receptors with altered properties, such as in autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and congenital myasthenic syndromes. A logical approach to treating diseases that involve a loss of cholinergic function in the CNS would be to attempt to restore this function. The use of full or partial agonists to activate nAChRs in the CNS is likely to cause chronic receptor inactivation. A more suitable method of restoring cholinergic function may be to increase the activity of the remaining receptors by allosteric potentiation. b-Amyloid peptides have been associated with neuritic plaques found in the brains of Alzheimer’s sufferers. The most abundant b-amyloid peptide is 40 amino acids long (Ab1–40); however, a small fraction of peptide is 42 amino acids in length (Ab1–42). Both of these peptides have been demonstrated to bind to and inhibit a7 nAChRs in the hippocampus (83,84). Conroy et al. demonstrated that inhibition of a7 receptors by b-amyloid peptide can be reversed by the positive allosteric modulator BSA (see section ‘‘Serum Albumin’’), indicating that treatment with positive allosteric modulators may represent a promising therapeutic strategy for the early treatment of Alzheimer’s disease. Mutations in the genes coding for the a4 and b2 subunits of the nAChR give rise to single-nucleotide polymorphisms, which modify several aspects of receptor behavior. A common feature of all the mutations associated with ADNFLE is an increased sensitivity to ACh, which results in a gain of receptor function (85–88). A desirable therapeutic strategy for the treatment of ADNFLE would aim to reduce this increased function, without causing widespread inhibition of a4b2 receptors. There is experimental evidence to suggest that mutant receptors have a pharmacology that is different from the wild-type receptor. An increased sensitivity to the antiepileptic drug carbamazepine was reported among some NFLE and ADNFLE sufferers, and in vitro a4-S248F and a4-L-776ins3 ADNFLE mutant receptors were more sensitive to inhibition by carbamazepine than wild-type a4b2 receptors [reviewed by Hogg and Bertrand (89)]. Carbamazepine causes open channel block of nAChRs; the increased sensitivity of the receptors containing the ADNFLE mutations indicates that these receptors present structurally

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distinct binding sites on their surface that are separate from the ACh binding site. The targeting of ligands to these sites may represent a promising approach to selective inhibition of ADNFLE mutant receptors. Advantages of Allosteric Modulators Traditionally, drugs that target membrane receptors are directed to the native ligand binding site, or in the case of some ion channels to the ionconducting pore. Targeting compounds toward the natural agonist binding site is the most obvious way to ﬁnd a molecule that acts selectively at the desired receptor, and permits easy screening of candidate molecules using competitive binding assays. As therapeutics, molecules that act allosterically to modify receptor properties have some advantages over those acting at the agonist binding site. For drugs designed to increase receptor function this has an important advantage over a full or partial agonist. As they exert their effects only when the natural agonist is present, only physiological responses are potentiated and chronic receptor activation with the associated problem of receptor desensitization is avoided. It is also possible using allosteric modulators to alter the time course of the response; compounds that change L1 will effect the transition of the receptor into the desensitized state and can either lengthen or shorten the response duration. 5-HI increases the peak response at a7 receptors without affecting the time course, whereas ivermectin causes a prolongation of the response duration (44,48), suggesting that ivermectin both lowers L0 and raises L1. For receptors that have a considerable permeability to Ca2þ, such as a7, a prolongation of the time course of response can have an important impact on the amount of Ca2þ entering the cell; since Ca2þ is involved in several intracellular signaling pathways, this can have an important impact on the functioning of the cell. Screening for Allosteric Modulators To screen potential drug candidates, most drug discovery programs employ a competitive binding assay such as a ﬂuorescent assay, for example, a ﬂuorometric imaging plate reader (FLIPR) or a comparable method, which involves competition with a labeled ligand, which binds to the natural agonist binding site. Allosteric ligands may or may not have effects on the afﬁnity of binding of a ligand to the natural ligand binding site. As in many cases, allosteric modulation will result in no detectable changes in binding afﬁnities; functional studies are indispensable for the detection and characterization of allosteric effectors. While high-throughput screening requires a multiwell assay such as FLIPR, it should be remembered that the activity of fast desensitizing receptors such as a7 cannot be presently detected by such assays. In addition, as the nicotinic receptor exhibits a marked voltage dependency and rectiﬁcation, the resting potential at which the assay is

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effectuated becomes the determining factor. It is therefore indispensable to design experiments in voltage clamp conditions that allow the maintenance of the transmembrane potential at a value corresponding to the range of activity of the nAChRs.

CONCLUSIONS The identiﬁcation of an entire family of genes that encode for neuronal nAChRs opened new questions about the function of these ligand-gated channels in the CNS. The importance of these neuroreceptors is supported by the widespread effects caused by the natural alkaloid nicotine as well as compounds that interact with the cholinergic system. It should also be remembered that these receptors have been associated with several neurological diseases, which suggests that they should constitute important therapeutic targets. While conventional agonists and antagonists obviously represent one possibility to interact with ligand-gated channels, allosteric effectors constitute another important alternative. The importance of allosteric modulation as an endogenous mechanism in the modulation of nAChRs is illustrated by the endogenous proteins Lynx-1 and SLURP-1, as well as steroid modulation and intracellular mechanisms such as phosphorylation. In addition, modulation of nAChRs by calcium and zinc have been demonstrated to be associated with synaptic transmission. In setting the goals for the future it should be kept in mind that allosteric effectors offer a unique advantage over conventional agonists and antagonists, offering the possibility to modulate the activity of a given protein, which remains under the control of the natural processes, a concept that becomes all the more relevant if we consider the importance of ﬁnetuning mechanisms that are necessary for the proper functioning of neuronal networks.

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55. Buisson B, Gopalakrishnan M, Arneric SP, Sullivan JP, Bertrand D. Human alpha4beta2 neuronal nicotinic acetylcholine receptor in HEK 293 cells: A patch-clamp study. J Neurosci 1996; 16(24):7880–7891. 56. Mulle C, Lena C, Changeux JP. Potentiation of nicotinic receptor response by external calcium in rat central neurons. Neuron 1992; 8(5):937–945. 57. Booker TK, Smith KW, Dodrill C, Collins AC. Calcium modulation of activation and desensitization of nicotinic receptors from mouse brain. J Neurochem 1998; 71(4):1490–1500. 58. Amador M, Dani JA. Mechanism for modulation of nicotinic acetylcholine receptors that can inﬂuence synaptic transmission. J Neurosci 1995; 15(6): 4525–4532. 59. Bonfante-Cabarcas R, Swanson KL, Alkondon M, Albuquerque EX. Diversity of nicotinic acetylcholine receptors in rat hippocampal neurons. IV. Regulation by external Caþþ of alpha-bungarotoxin-sensitive receptor function and of rectiﬁcation induced by internal Mgþþ. J Pharmacol Exp Ther 1996; 277(1):432–444. 60. Galzi JL, Bertrand S, Corringer PJ, Changeux JP, Bertrand D. Identiﬁcation of calcium binding sites that regulate potentiation of a neuronal nicotinic acetylcholine receptor. EMBO J 1996; 15(21):5824–5832. 61. Swope SL, Moss SJ, Blackstone CD, Huganir RL. Phosphorylation of ligandgated ion channels: a possible mode of synaptic plasticity. FASEB J 1992; 6(8):2514–2523. 62. Wecker L, Guo X, Rycerz AM, Edwards SC. Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits. J Neurochem 2001; 76(3):711–720. 63. Albuquerque EX, Deshpande SS, Aracava Y, Alkondon M, Daly JW. A possible involvement of cyclic AMP in the expression of desensitization of the nicotinic acetylcholine receptor. A study with forskolin and its analogs. FEBS Lett 1986; 199(1):113–120. 64. Middleton P, Jaramillo F, Schuetze SM. Forskolin increases the rate of acetylcholine receptor desensitization at rat soleus endplates. Proc Natl Acad Sci USA 1986; 83(13):4967–4971. 65. Middleton P, Rubin LL, Schuetze SM. Desensitization of acetylcholine receptors in rat myotubes is enhanced by agents that elevate intracellular cAMP. J Neurosci 1988; 8(9):3405–3412. 66. Mulle C, Benoit P, Pinset C, Roa M, Changeux JP. Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells. Proc Natl Acad Sci USA 1988; 85(15): 5728–5732. 67. Huganir RL. Regulation of the nicotinic acetylcholine receptor by serine and tyrosine protein kinases. Adv Exp Med Biol 1991; 287:279–294. 68. Wagner K, Edson K, Heginbotham L, Post M, Huganir RL, Czernik AJ. Determination of the tyrosine phosphorylation sites of the nicotinic acetylcholine receptor. J Biol Chem 1991; 266(35):23, 784–23,789. 69. Fenster CP, Beckman ML, Parker JC, et al. Regulation of alpha4beta2 nicotinic receptor desensitization by calcium and protein kinase C. Mol Pharmacol 1999; 55(3):432–443.

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70. Nishizaki T, Matsuoka T, Nomura T, Sumikawa K. Modulation of ACh receptor currents by arachidonic acid. Brain Res Mol Brain Res 1998; 57(1): 173–179. 71. Wang K, Hackett JT, Cox ME, Van Hoek M, Lindstrom JM, Parsons SJ. Regulation of the neuronal nicotinic acetylcholine receptor by SRC family tyrosine kinases. J Biol Chem 2004; 279(10):8779–8786. 72. Miwa JM, Ibanez-Tallon I, Crabtree GW, et al. lynx1, an endogenous toxinlike modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 1999; 23(1):105–114. 73. Ibanez-Tallon I, Miwa JM, Wang HL, et al. Novel modulation of neuronal nicotinic acetylcholine receptors by association with the endogenous prototoxin lynx1. Neuron 2002; 33(6):893–903. 74. Mastrangeli R, Donini S, Kelton CA, et al. ARS Component B: structural characterization, tissue expression and regulation of the gene and protein (SLURP-1) associated with Mal de Meleda. Eur J Dermatol 2003; 13(6):560–570. 75. Maus AD, Pereira EF, Karachunski PI, et al. Human and rodent bronchial epithelial cells express functional nicotinic acetylcholine receptors. Mol Pharmacol 1998; 54(5):779–788. 76. Arredondo J, Nguyen VT, Chernyavsky AI, et al. Central role of alpha7 nicotinic receptor in differentiation of the stratiﬁed squamous epithelium. J Cell Biol 2002; 159(2):325–336. 77. Arredondo J, Nguyen VT, Chernyavsky AI, et al. Functional role of alpha7 nicotinic receptor in physiological control of cutaneous homeostasis. Life Sci 2003; 72(18–19):2063–2067. 78. Chimienti F, Hogg RC, Plantard L, et al. Identiﬁcation of SLURP-1 as an epidermal neuromodulator explains the clinical phenotype of Mal de Meleda. Hum Mol Genet 2003; 12(22):3017–3024. 79. Di Angelantonio S, Costa V, Carloni P, Messori L, Nistri A. A novel class of peptides with facilitating action on neuronal nicotinic receptors of rat chromafﬁn cells in vitro: functional and molecular dynamics studies. Mol Pharmacol 2002; 61(1):43–54. 80. Di Angelantonio S, Giniatullin R, Costa V, Sokolova E, Nistri A. Modulation of neuronal nicotinic receptor function by the neuropeptides CGRP and substance P on autonomic nerve cells. Br J Pharmacol 2003; 139(6):1061–1073. 81. Weiland S, Bertrand D, Leonard S. Neuronal nicotinic acetylcholine receptors: from the gene to the disease. Behav Brain Res 2000; 113(1–2):43–56. 82. Graham AJ, Martin-Ruiz CM, Teaktong T, Ray MA, Court JA. Human brain nicotinic receptors, their distribution and participation in neuropsychiatric disorders. Curr Drug Targets CNS Neurol Disord 2002; 1(4):387–397. 83. Liu Q, Kawai H, Berg DK. Beta-amyloid peptide blocks the response of alpha 7-containing nicotinic receptors on hippocampal neurons. Proc Natl Acad Sci USA 2001; 98(8):4734–4739. 84. Pettit DL, Shao Z, Yakel JL. Beta-amyloid(1–42) peptide directly modulates nicotinic receptors in the rat hippocampal slice. J Neurosci 2001; 21(1):RC120. 85. Picard F, Bertrand S, Steinlein OK, Bertrand D. Mutated nicotinic receptors responsible for autosomal dominant nocturnal frontal lobe epilepsy are more sensitive to carbamazepine. Epilepsia 1999; 40(9):1198–1209.

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86. Phillips HA, Favre I, Kirkpatrick M, et al. CHRNB2 is the second acetylcholine receptor subunit associated with autosomal dominant nocturnal frontal lobe epilepsy. Am J Hum Genet 2001; 68(1):225–231. 87. Rodrigues-Pinguet NO, Jia L, Li M, et al. Mammalian ﬁve ADNFLE mutations reduce Ca2þ dependence of the {alpha}4{beta}2 acetylcholine response. J Physiol 2003; 18:18. 88. Hogg RC, Bertrand D. Regulating the regulators: the role of nicotinic acetylcholine receptors in human epilepsy. Drug News Perspect 2003; 16(5):261–266. 89. Hogg RC, Bertrand D. Neuronal nicotinic receptors and epilepsy, from genes to possible therapeutic compounds. Bioorg Med Chem Lett 2004; 14(8):1859–1861.

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SECTION III: G-PROTEIN–COUPLED RECEPTORS

8 Allosteric Modulation of G-Protein–Coupled Receptors Willem Soudijn, Ineke van Wijngaarden, and Ad P. Ijzerman Division of Medicinal Chemistry, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands

INTRODUCTION G-protein–coupled receptors (GPCRs) constitute a very important class of drug targets. It is estimated that approximately half of the marketed medicines exert their action through members of this protein class (1). These membrane-bound macromolecules most probably share the architecture of the visual pigment rhodopsin, i.e., a cavity with seven transmembrane a-helical domains as outer boundaries, ideally suited to accommodate small molecules or molecular fragments. Such molecules are the endogenous agonists in the ﬁrst place, i.e., hormones and neurotransmitters. They act as ‘‘primary messengers’’ to convey signals into the cell that give rise to, among others, the production of ‘‘second messengers’’ such as cyclic adenosine monophosphate (cAMP), leading to a cellular response. Synthetic agonists, antagonists, and inverse agonists have been introduced to the market, yielding the rich repertoire of current medicines. Careful reﬂection, however, teaches that the receptor interaction of hormones and neurotransmitters is quite different from most synthetic drugs. These natural ligands are often synthesized in situ on demand. They may also be subject to rapid and extensive metabolism, often at the site of action. Both processes of synthesis and breakdown effectively cause a

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transient receptor stimulation. In contrast, synthetic agonists (such as morphine or isoproterenol) are often metabolically stable. This may lead to a more continuous stimulation of receptor proteins, which is not necessarily desirable. Similarly, the receptor occupancy by an antagonist can yield a prolonged blockade of receptor activity, which may be very different from the ‘‘kinetics’’ of a disease state. Can this be done in a more sophisticated way? The answer is afﬁrmative, in that a more controlled and selective ‘‘tuning’’ action on the receptor is feasible through allosteric modulation. In enzyme research this phenomenon is well known and has long been recognized as a general mechanism for the control of protein function (2). ‘‘Allosteric’’ or ‘‘allotopic’’ refers to binding sites different from the ‘‘orthosteric’’ primary substrate or ligand binding site. If ligands bind to such allosteric sites, a conformational change of the receptor protein may occur, which in turn may inﬂuence GPCR ligand binding and function. Since the endogenous ligand remains to play a key role, the overall pharmacology resembles physiology more closely than with the use of synthetic ligands (Fig. 1). The purpose of this chapter is to review the latest ﬁndings in this fascinating ﬁeld of research. This includes, apart from a limited discussion of representatives of the current ligand repertoire, an exploration of allosteric binding sites mainly from receptor mutation studies, available clinical data, and a reﬂection on the deﬁnition of allosteric modulation. In addition, we would like to make a plea for re-engineering some of the available receptor assays to screen for allosteric modulation.

Figure 1 An allosteric enhancer offers a more physiologic alternative to synthetic agonists. It can only act in the presence of an (endogenous) agonist, which mimics the duration and intensity of action of hormone or neurotransmitter much better than that induced by a synthetic agonist (see the three panels). This combined action of two compounds (i.e., agonist and enhancer) may also induce signiﬁcant gains in selectivity of drug action.

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SPECIFIC EXAMPLES OF ALLOSTERIC MODULATORS In this paragraph we will speciﬁcally address the interaction of small molecules with the receptor macromolecule itself rather than mention other allosteric interactions, such as with G-proteins. Therefore, we will focus on small organic molecules that have been shown to interact with the receptor protein in an allosteric fashion and that may be regarded as lead candidates for future medicines. With respect to receptor molecules we follow the subdivision of human receptors into classes 1 (or A, rhodopsin-like), 2 (or B, secretin-like), and 3 (or C, metabotropic glutamate–like) (3). The scope of this review is limited in that only some recent examples and literature can be examined. Therefore, we also refer the interested reader to a number of other reviews on the same subject (4–6). Class 1 Receptors The rhodopsin-like receptors constitute by far the largest group of GPCRs, termed class 1. Many representatives have been shown to be regulated allosterically. The most extensive studies have been performed on adenosine and muscarinic receptors, which will be discussed in this section. Allosteric Modulators of Adenosine Receptors The ﬁrst allosteric modulator of the adenosine A1 receptor was described by Bruns et al. (7,8). They discovered that several 2-amino-3-benzoylthiophenes selectively enhance the binding and function of reference A1 receptor agonists. The most interesting compound of the series is PD81,723 (2-amino-4,5-dimethyl-3-thienyl)[(3-triﬂuoromethyl)phenyl] methanone (Fig. 2). PD81,723 is effective in various species, including human. Allosteric modulation has been demonstrated in in vitro and in vivo studies, e.g., on cloned human A1 adenosine receptors (9) and in a dog model of cardiac ischemia (10). Long-term exposure of intact Chinese hamster ovary (CHO)-K1 cells stably expressing the human A1 adenosine receptor to

Figure 2 Structures of reference allosteric enhancers of the adenosine A1 receptor.

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PD81,723 produced no desensitization with regard to its allosteric action (11). At higher concentrations, PD81,723 acts as an inhibitor of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a well-known antagonist of the adenosine A1 receptor. Currently, DPCPX has been classiﬁed as an inverse agonist of A1 receptors (12). Several groups extended the structure–activity relationship (SAR) studies of Bruns. Using PD81,723 as the lead compound, IJzerman et al. showed that in the dialkyl and in the cyclized PD81,723 analogs an appropriately substituted benzoyl ring is essential for high activity (Table 1) (13). Favorable substituents are 4-I, 3,4-Cl, 4-CH3, 4-CF3, 4-Br, 4-Cl, and 3-CF3. A 4-NO2 substituent is not tolerated. Similar to PD81,723, all analogs act as antagonists of the inverse agonist DPCPX. However, the SAR for antagonism and allosteric enhancement is distinct (Table 1). The most promising compound of the series is LUF5484 (2amino-3-(3,4-dichlorobenzoyl)-4,5,6,7-tetrahydrobenzo[b]thiophene), being 2.4-fold more potent than PD81,723 as an enhancer, while showing comparable antagonistic activity (Table 1). In the series of 6-benzyl-2amino-3-benzoyl-4,5,6,7-tetrahydrothieno(2,3-c) pyridines a 3,4-dichloro– substituted benzoyl moiety is similarly favorable for allosteric activity (13). The SAR of the 2-aminothiophenes on cloned human adenosine A1 receptors has been presented by Tranberg et al. (14). For the human adenosine A1 receptor too, an appropriately substituted benzoyl group and substitution at C-4 and C-5 with alkyl or a polymethylene bridge is essential for allosteric activity. The rank order of potency is cHep cHex > cPent 4,5-Me 4,5-H. Favorable 3-benzoyl substituents are, e.g., 4-I, 4-Br, 4-OCH3, 4-cHex, 3-I, 3-Br, and 3-OCH3. The lack of antagonistic activity of the potent allosteric enhancers belonging to the cycloheptylthiophenes is striking. The corresponding cyclopentyl and cyclohexyl analogs act as antagonists of the inverse agonist DPCPX (Table 2). Substitution at C-4 and C-5 with aryl groups is detrimental to allosteric activity (15). The 4-aryl-5-bromothiophenes, however, show substantial enhancing activity. The most potent compound of the series is [2-amino-5-bromo-4-(3-triﬂuoromethylphenyl)thiophen-3-yl]phenylmethanone. The compound is ﬁve times more active than PD81,723 as an allosteric enhancer, while showing threefold less antagonistic activity. The effective concentration (EC50) values are similar: 8.4 and 6.4 mM, respectively. A novel chemical class of allosteric enhancers of agonist binding at cloned human A1 adenosine receptor are the 2-aminothiazoles (16). Table 3 shows that a planar or nearly planar tricyclic ring system is essential for allosteric enhancing activity. The rank order of potency is 6:5:5 6:6:5 6:7:5. Substitution at the four-position of the indenothiazole ring with acetyl enhances the activity 127-fold (EC50 values: 0.3 and 38 mM, respectively). The corresponding cyclopentyl ester is less potent (EC50 ¼ 4.5 mM). The cyclohexyl ester displays no allosteric enhancing activity. Other favorable substituents are 4-CH3, 6-CH3, and 5,6-OCH3. Weak enhancers are

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Table 1 Adenosine A1 Receptor Enhancement and Antagonism by 2-Aminobenzoylthiophenes in Rat Brain Membranes

Compound

R0

PD 81,723

3-CF3 H 3-Cl 4-Cl H 2-Cl 3-Cl 4-Cl 3-I 3-CF3 4-Br 4-I 4-CF3 4-CH3 4-NO2 3,4-Cl

PD 71,605 T62

LUF5484

R4

R5

CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4– –(CH2)4–

Enhancement Enhancement (%)a EC50 (mM) 100 8 80 94 47 73 93 123 113 122 128 155 131 137 34 151

14.7

10.5 6.8 6.0 16.4 4.5 4.7 34.9 6.2

Antagonism (%)b 40 14 19 41 35 35 51 40 66 32 42 64 57 30 20 35

a

Enhancing activity by 10 mM of test compound is expressed as percent decrease in [3H]CCPA (2-chloro-N6-cyclopentyladenosine) dissociation over control. b Antagonistic activity is expressed as percent displacement of 0.4 nM [3H]DPCPX by 10 mM of test compound. Abbreviations: CCPA, 2-chloro-N6-cyclopentyl adenosine; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine.

compounds substituted with 5-OCH3 or 4,5,6-OCH3. The corresponding 5Br analog is inactive. In the 6:6:5 series the rank order of potency is acetyl > methoxy > hydrogen (EC50 values: 3.8, 8.7, and 17 mM, respectively). The position isomer 10-H-indeno(1,2-d) thiazol-2-yl-amine is devoid of allosteric enhancing activity. This loss in activity may be due to steric hindrance of the phenyl group.

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Table 2 Adenosine A1 Receptor Enhancement and Antagonism by 2-Aminobenzoylthiophenes in Membranes from CHO-K1 Cells Expressing the Human Adenosine A1 Receptor

Compound PD 81,723

R H 3-CF3 H 3-Br 4-Br H 3-Cl 4-Br 4-I 4-cHex H 3-Br 3-OCH3 4-Br 4-I

n

Enhancement (%)a

Antagonism (%)b

0 0 1 1 1 2 2 2 2 2 3 3 3 3 3

9 19 20 68 62 19 70 83 86 99 13 78 99 86 96

42 56 36 20 42 78 61 62 47 68 7 13 7 9

a

Enhancing activity by 100 mM of test compound is expressed as percent decrease in [125I]ABA dissociation over control. b Antagonistic activity is expressed as percent displacement of 4 nM [3H]DPCPX by 100 mM of test compound. Abbreviations: CHO, Chinese hamster ovary; ABA, N6-p-aminobenzyladenosine; DPCPX, 1,3dipropyl-8-cyclopentylxanthine.

The allosteric enhancer of adenosine A1 receptors T62 (Table 1) reduces the hypersensitivity in animal models of chronic pain (17). T62 has been tested on a limited number of healthy volunteers for dose ﬁnding (18). The results of this initial clinical phase 1 trial have not been reported yet. The ﬁrst selective allosteric enhancer of agonist binding at human A3-adenosine (A3A) receptors is VUF5455, a 3-(2-pyridinyl) isoquinoline derivative (Fig. 3) (19). In a cAMP second messenger assay the effects of Cl-IB-MECA on forskolin-induced cAMP formation were signiﬁcantly

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Table 3 Adenosine A1 Receptor Enhancement by 2-Aminothiazoles in Membranes from CHO-K1 Cells Stably Expressing the Human Adenosine A1 Receptor

Compound

R

N

H H H 4-CH3 4-OCOCH3 4-OCOC5H9 4-OCOC6H11 5-OCH3 5-OCH3 5-Br 5-OCOCH3 6-CH3 5,6-OCH3 4,5,6-OCH3 5,6,7-OCH3

1 2 3 1 1 1 1 1 2 1 2 1 1 1 1

PD 81,723

Enhancement (%)a

Enhancement EC50 (mM)

91 80 n.a. 81 80 85 n.a. 66 94b n.a. 92b 72 99b 60 24 50b

38 17 n.d. 0.3 4.5 50 8.7 3.8 4.2 1.2 17 n.d. 38

a

Enhancing activity by 50 mM of test compound is expressed as percent decrease in [125I]ABA dissociation over control. b Enhancing activity by 100 mM of test compound is expressed as percent decrease in [125I]ABA dissociation over control. Abbreviations: CHO, Chinese hamster ovary; ABA, N6-p-aminobenzyladenosine; n.a., not applicable; n.d., not determined.

enhanced by VUF5455 (EC50 values: 232 and 72.5 nM, respectively). Substitution of hydrogen for 7-CH3 (VUF8504) or for 4-OCH3 and 7-CH3 (VUF8507) does not affect the allosteric enhancing activity. However, the compounds display different afﬁnities for the human A3A receptor. The Ki values are 1680 nM (VUF5455), 17.3 nM (VUF8504), and 204 nM (VUF8507). These results show that VUF5455 can be used as a lead structure for the design of pure allosteric enhancers of A3A receptors. A novel structural lead for developing allosteric enhancers of A3A receptors is Du 124183, an imidazoquinoline derivative (Fig. 3) (20). Du 124183 selectively enhances the

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Figure 3 Allosteric modulators of adenosine A3 receptors.

agonist binding and function at human A3A receptors. The compound has a unique mechanism of action as it enhances the maximum efﬁcacy of A3A receptors in the cAMP assay by 30%. In competitive binding studies Du 124183 binds with moderate afﬁnity (Ki ¼ 820 nM). Mutagenesis studies in COS-7 cells transiently expressing the wild-type adenosine A3 receptor and its mutants show that a number of amino acids appear to be involved in the recognition of the allosteric modulators (21). The mutations Asn30Ala (TM 1), Asp58Asn (TM 2), Phe182Ala (TM 5), and Asn274Ala (TM7) abolish the ability of VUF5455 and Du 124183 to decrease the dissociation rate of 125 I-AB-MECA. Interestingly, the Asp107Asn mutation is detrimental to Du 124183 but not to VUF5455. Allosteric Modulators of Muscarinic Receptors Double ring closure of alcuronium yielded diallylcaracurine V (Fig. 4). It is structurally closely related to alcuronium but has two additional asymmetric centers in its structure (22). Diallylcaracurine V showed a high afﬁnity for M2 receptors similar to that of alcuronium (23) and a high selectivity versus M5 receptors (24). In search of binding sites at the M2 receptor for the allosteric ligands diallylcaracurine V and W84 (Fig. 4), point mutants of human M2 and M5 receptors were constructed and, like the wild-type receptors, transiently expressed in COS-7 cells (24,25). Regions in the second outer loop and at the beginning of TM 7 of the receptors were considered to be involved in binding and selectivity of the allosteric compounds (24). A likely amino acid candidate that forms part of a binding site for the allosteric compounds is Thr423. This stems from the observation that a high

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Figure 4 Allosteric modulators of muscarinic acetylcholine receptors. (Continued on next page.)

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Figure 4 (Continued from previous page.)

degree of conservation of corresponding amino acids exists at the end of the third outer loop and the beginning of TM 7 in M2 and M5 receptors, the only difference in amino acids being threonine in position 423 of M2 receptors (Thr423) and histidine (His478) in the corresponding position of the M5 receptor. In the middle region of the second outer loop of the M2 receptor, tyrosine 177 (Tyr177) was also shown to be involved in the binding and selectivity of the allosteric agents, whereas in the corresponding position of the M5 receptor, glutamine 184 (Gln184) is present (25). Point mutants of M2 receptors were constructed by replacement of Tyr177 or Thr423 by the corresponding amino acids of M5 receptors Gln184 or His478, respectively, and double point mutants by replacement of both Tyr177 and Thr423 by the pair of Gln184 and His478. The potency of the allosteric compounds in reducing the rate of dissociation of the radioligand [3H]NMS (N-methylscopolamine) from wild-type and mutant receptors was expressed as pEC0.5,diss, a parameter representing the modulator concentration at which the dissociation rate decreases to half of its control value. The pEC0.5,diss values of diallylcaracurine, W84, and dimethyl-W84 using wild-type M2 receptors were 8.00, 7.42, and 8.09, respectively. With wild-type M5 receptors these values were 5.69, 5.54, and 5.78, respectively. For the M2 double point mutant (Tyr177 ! Gln184 and Thr423 ! His478) receptors these values were 5.72, 5.54, and 6.02, respectively, very similar to the wild-type M5 receptor values. The pEC0.5,diss values of the allosteric compounds using M2 receptor single point mutants Tyr177 ! Gln184 or Thr423 ! His478 were also shifted signiﬁcantly toward the pEC0.5,diss values obtained using wild-type M5 receptors (25). The effect of a series of allosteric modulators on the binding of [3H]NMS to M2 receptors in the membranes of pig heart ventricles

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was described by Muth et al. (26). The series consisted of W-84, 5-dimethylW-84, di-1,8-naphthyl analogs of W-84, the 2,2-dimethylpropyl derivatives, and hybrids (Fig. 4). The afﬁnity of the modulators for free M2 receptors (pKA) was compared with the afﬁnity for [3H]NMS-occupied receptors p(aKA), where a ¼ cooperativity factor. If p(aKA) < pKA the cooperativity with [3H]NMS is deﬁned to be negative. Allosteric W-84 showed negative cooperativity between W84 and [3H]NMS binding, as p(aKA) 5.73 < pKA 6.19. Introduction of one 2,2dimethylpropyl group at the C-2 of W84 (Fig. 4) resulted in neutral cooperativity as p(aKA) and pKA were similar: 6.90 and 6.86, respectively. However, introduction of a second 2,2-dimethylpropyl group at the C-2 of W84 (Fig. 4) reversed the cooperativity from neutral to negative again: p(aKA) 6.87 < pKA 7.08. Negative cooperativity comparable with that of W84 was shown by 5-dimethyl-W84, as p(aKA) 6.75 < pKA 7.08, and neutral cooperativity by introduction of one 2,2-dimethylpropyl group at the C-2 of 5-dimethyl-W84: p(aKA) 7.41 and pKA 7.44. Introduction of a second 2,2dimethylpropyl group at the C-20 of 5-dimethyl-W84, however, changed the neutral cooperativity to a weak positive one: p(aKA) 7.38 > pKA 7.21. Replacement of both phthalimido groups in W84 by 1,8 naphthalimido groups (Fig. 4) yielded an allosteric modulator negatively cooperating with [3H]NMS binding, as p(aKA) 7.83 < pKA 8.11. In this case introduction of one 2,2-dimethylpropyl group at C-2 caused not a neutral but a modestly positive cooperativity: p(aKA) 8.24 versus pKA 8.07. A substantial positive cooperativity was obtained by introduction of a second 2,2-methylpropyl group at the C-20 of this compound: p(aKA) 8.04 versus pKA 7.51. Replacement of one naphthalimido group by a phthalimido group yielded a hybrid napht/phtal compound (Fig. 4) with negative cooperativity: p(aKA) 7.04 < pKA 7.24. Positive cooperativity [p(aKA) 8.66 > pKA 8.29] was obtained by replacement of the propyl group on the napht side of the molecule by a 2,2-dimethylpropyl group. However, replacement on the phtal side of the molecule resulted in neutral cooperativity: p(aKA) ¼ pKA ¼ 7.28. Replacement of the propyl group by 2,2-dimethylpropyl on both sides of the napht/phtal hybrid resulted in positive cooperativity by the modulator: p(aKA) 7.97 > pKA 7.41, which was comparable to that of the 1.8 naphthalimido parent compound: p(aKA) 8.04 > pKA 7.51. A new enhancer of acetylcholine binding to muscarinic M4 receptors was recently reported by Lazareno et al. (27). Thiochrome (Fig. 4) at 100 mM increased the afﬁnity of acetylcholine fourfold for inhibiting [3H]NMS binding to this receptor subtype. Similarly, in [35S]GTPcS binding assays the compound enhanced the potency of acetylcholine on M4, but not on M1, M2, and M3, receptors. The compound was also active in a more functional assay, as it reduced acetylcholine release from rat striatal slices containing autoinhibitory presynaptic M4 receptors but not from hippocampus (M2 receptors). For this compound the authors did not hesitate

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to use the term ‘‘absolute subtype selectivity,’’ as a new from of selectivity based on cooperativity rather than afﬁnity. The discovery of ‘‘allosteric agonists’’ for muscarinic receptors has been recently reported by independent research groups. Such compounds appear to activate the receptor at sites distant from the acetylcholine binding site. They are not true modulators, in that their activity does not require the presence of the endogenous agonist. AC-42 (Fig. 4), a lead compound resulting from a broad screening program, is a typical example (28). It was demonstrated to behave as an agonist for the muscarinic M1 receptor. By making chimeras between the muscarinic M1 and the M5 receptor, the authors provided proof of the compound interacting among others with parts of the N-terminus and the upper region of TM1 in the M1 receptor. Similarly, N-desmethylclozapine (Fig. 4) was identiﬁed as an allosteric muscarinic M1 receptor (partial) agonist (29). The compound penetrates the brain and is able to potentiate hippocampal N-methyl-D-aspartic acid receptor currents through M1 receptor activation. A tyrosine residue in TM6 (Tyr381) appeared to play an important role in the binding process, and was suggested to form part of this distant binding site. When this residue was mutated to alanine, the receptor largely lost its capability of recognizing acetylcholine, whereas N-desmethylclozapine turned into a very potent full agonist. This ﬁnding led the authors to conclude that the natural acetylcholine binding site does not fully overlap the one for N-desmethylclozapine. Class 2 Receptors The receptors in this family are targets for large endogenous peptides with high amino acid identity. The secretin receptor was the ﬁrst one to be cloned in this family, hence the often used term of ‘‘secretin-like’’ receptor class. The receptor N-terminus, with a length of 60–80 amino acids, contains conserved cysteine bridges and is particularly important for the binding of the natural peptide ligands. Other receptors with even longer N-terminal tails (up to several times the size of the TM domain) may be regarded as a subclass of class 2 receptors, although this is subject to debate. These tails contain a number of well-characterized protein modules with, e.g., immunoglobulin motifs [for a review see (30)]. In these cases the endogenous ligands and details of their binding sites are often unknown. There is limited, though convincing, evidence for allosteric modulation of class 2 receptors, of which we will give two examples. Allosteric Modulators of Corticotropin-Releasing Factor Type Receptors The nonpeptide antagonists (NPAs) antalarmin, DMP-696, NBI 27914 (Fig. 5), and NBI 35965 (chemical structure not disclosed) are potent and efﬁcacious allosteric inhibitors of peptide agonist binding to cloned

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Figure 5 Allosteric modulators of corticotropin-releasing factor type 1 (CRF1) receptors.

human corticotropin-releasing factor (CRF1) receptors stably expressed in Ltk-cells (31). NBI 35965 slowed down the dissociation of 125ICRF and [125I]sauvagine from the receptor in a concentration-dependent way with EC50 values of 52 and 130 nM, respectively. The NPAs display similar potency in displacing [125I]sauvagine binding: antalarmin (Ki ¼ 0.38 nM), NBI 27914 (Ki ¼ 0.95 nM), NBI 35965 (Ki ¼ 1.45 nM), and DMP-696 (Ki ¼ 2.34 nM). Speciﬁc [3H]NBI 35965 binding was fully inhibited by all NPAs, indicating that the NPAs bind to the same site on the CRF1 receptor.

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The extracellular N-terminal domain (CRF1-N) (residues 1–118) and the juxtamembrane domain of the CRF receptor (CRF1-J) (residues 110–415) have been expressed independently by Hoare et al. (32). The NPAs do not bind to CRF1-N but display high afﬁnity for CRF1-J. The Ki values for displacing [3H]NBI 35965 binding to CRF1-J and to wild-type (WT) CRF1 receptors are the same: antalarmin 1.1 nM (CRF1-J), 1.3 nM (WT); DMP696 1.9 nM (CRF1-J and WT); NBI 27914 2.8 nM (CRF1-J), 1.6 nM (WT); and NBI 35965 1.8 nM (CRF1-J), 2.1 nM (WT). The peptide agonists CRF and sauvagine displayed similar afﬁnities for the CRF1-N and CRF1-WT (Ki values of 56 and 51 nM for CRF, and 490 and 140 nM for sauvagine, respectively). These results show that the NPAs bind only to the J-domain whereas the peptide agonists bind to the N-terminal domain. NPAs for the CRF1 receptor may be useful in the treatment of CRF-associated disorders such as stress and depression. R-121919 (Fig. 5) has been tested in patients with major depression. The compound ameliorated depressive symptoms without undesired endocrine side effects. Further development of R-121919 has been discontinued after phase IIa studies. At present, a number of other CRF1 receptor antagonists have entered clinical trials. The results of these studies have not been published yet (33). Allosteric Modulators of Glucagon and Glucagon-Like Peptide-1 Receptors Glucagon, a peptide hormone 29 amino acids long, activates the G-protein– coupled glucagon receptor resulting in increased glycogenolysis and gluconeogenesis causing a net rise of glucose levels in the blood. Antagonists of the glucagon receptor could help in diminishing the blood glucose level in type 2 diabetic patients. From a large series of potentially useful antagonists reported by Madsen et al. (34), one compound (NNC 25–2504, Fig. 6), a

Figure 6 Allosteric modulators of the glucagon and glucagon-like peptide (GLP-1) receptors.

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noncompetitive inhibitor of human glucagon receptors expressed in baby hamster kidney (BHK) cells, was chosen for further pharmacological research. NNC 25–2504 inhibited the binding of [125I]glucagon to human glucagon receptors in the membranes of BHK cells with an inhibitory concentration 50% (IC50) value of 2.3 nM. Stimulation of the human glucagon receptor by glucagon leads to an increase in cAMP production, which was competitively inhibited by 25 nM and noncompetitively by 250 and 2500 nM of NNC 25–2504. The KB value for this effect was 0.76 nM. In the same functional test using rat liver membranes NNC 25–2504 at concentrations of 1, 10, and 100 nM appeared to be a potent and noncompetitive inhibitor with a KB value of 0.38 nM. Glucagon administered to Sprague–Dawley rats induced a hyperglycemic effect that was abolished after a 10 mg/kg dose of NNC 25–2504. Glucagon-like peptide-1 (GLP-1) hormone, consisting of 30–31 amino acids, activates G-protein–coupled GLP-1 receptors resulting in, among other effects, potentiation of glucose-induced insulin secretion from pancreas b-cells and inhibition of glucagon secretion from pancreas a-cells (35, and references therein). The maintenance of a balanced glucose level in blood is thus mediated by the regulatory peptides glucagon and GLP-1. The molecular pharmacological properties of a noncompetitive GLP-1 receptor antagonist (T-0632, Fig. 6) were reported by Tibaduiza et al. (36). The afﬁnity of T-0632 for human and rat GLP-1 receptors expressed in COS-7 cells was assayed in competition binding experiments with the antagonist [125I]exendin(9-39), a peptide with a high afﬁnity for GLP-1 receptors. The afﬁnity of T-0632 for human or rat GLP-1 receptors expressed in IC50 values was 1.2 or 90 mM, respectively, indicating a large species selectivity of T-0632, whereas exendin(9-39) was not species selective. Binding experiments with GLP-1 receptor mutants showed that the tryptophan residue (Trp33) in the extracellular N-terminal domain of the human GLP-1 receptor was essential for the species selectivity of T-0632. Replacing the corresponding serine residue of the rat GLP-1 receptor by Trp33 yielded a mutant with characteristics of the human GLP-1 receptor with a comparable IC50 value of 0.7 mM for T-0632. Replacing Trp33 of the human GLP-1 receptor by serine yielded a mutant with characteristics of the rat GLP-1 receptor with an IC50 value of 143 mM for T-0632. cAMP production induced by GLP-1 activation of human and rat GLP-1 receptors was antagonized by T-0632 with IC50 values of 21 and 353 mM, respectively. T-0632 acted as a noncompetitive antagonist as the maximum cAMP production caused by GLP-1 was reduced by 23% in the presence of a ﬁxed concentration of T-0632 (2.3 104 M). Class 3 Receptors Class 3 receptors are characterized by a large N-terminal domain with specialized motifs (Venus ﬂy trap, VFT) that contain the actual neurotransmitter/

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hormone binding site, as is the case for c-aminobutyric acid (GABAB) receptors, glutamate (metabotropic glutamate receptors, mGluRs), and calcium ions (calcium-sensing receptors). We will discuss all three in more detail, also in view of recent clinical data for both metabotropic glutamate and calciumsensing receptors. Allosteric Modulators of GABAB Receptors GABAB receptors are heterodimers consisting of two subunits GB1 and GB2, both with a large extracellular domain containing a Venus ﬂy trap module (VFTM), seven transmembrane helices, and a large intracellular carboxyl terminal. Each VFTM is folded into a bilobate structure. The VFT of GB1 binds GABA and other selective agonists. The binding is enhanced by GB2 acting as a positive allosteric modulator. GB2 does not bind GABA-like agonists (37). The results of the ﬁrst allosteric modulator (CGP7930, Fig. 7) enhancing binding and efﬁcacy of GABA in activating GABAB receptors were published by Urwyler et al. in 2001 (38). The enhancing effect of CGP7930 on the inhibitory and stimulating properties of GABA receptor activation was described by Onali et al. (39). Basal adenylyl cyclase in membranes of the granule cell layer of the rat olfactory bulb was stimulated by GABA and this activity was enhanced by CGP7930. The pEC50 value of GABA for the stimulation of the basal cAMP production was 3.34. The enhancement in potency of GABA by 30 and 100 mM of CGP7930 resulted in pEC50 values of 3.36 and 4.02, respectively, and in enhancement of maximal cAMP formation by 29% and 49%. Corticotropin-releasing hormone stimulates the production of cAMP in membranes of rat frontal cortex, which is enhanced by GABA (pEC50 3.01) and signiﬁcantly increased by CGP7930 at 30 and 100 mM, resulting in pEC50 values of 3.37 and 3.76, respectively. The maximal effect was enhanced by 49% and 79%, respectively. Stimulation of adenylyl cyclase by forskolin was inhibited by

Figure 7 Allosteric modulators of c-aminobutyric acid (GABAB) receptors.

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activation of the GABAB receptor by GABA with a pEC50 value of 3.07. Again, the potency of GABA was enhanced, pEC50 3.29 and 3.85, by 30 and 100 mM of CGP7930, respectively. A series of novel structurally related allosteric enhancers of GABAB receptor function was described by Urwyler et al. (40). Chimeric GABAB1b/2 receptors – consisting of human GB1b and rat GB2 subunits – were stably expressed in CHO cells and membranes of the cells or native rat cortical brain cell membranes were used in the experiments. All compounds tested at 1 or 10 mM enhanced allosterically the effect of 1 mM of GABA activating GABAB1b/2 receptors to stimulate [35S]GTPcS binding to membranes of the CHO cells. The largest enhancement was caused by compound GS 39783 (Fig. 7). The effective concentrations (EC50 values) of GS 39783 for enhancing the effects of 1 or 20 mM of GABA on [35S]GTPcS binding to the recombinant CHO cell membranes were 3.5 and 2.1 mM, respectively. The EC50 values using rat cortical cell membranes were similar, 3.1 and 2.2 mM, respectively. In the presence of 30 mM of GS 39783 both potency and efﬁcacy of GABA in stimulating [35S]GTPcS binding to membranes of CHO cells expressing GABAB1b/2 receptors were maximally enhanced. In comparison with control data in the absence of GS 39783 the potency of GABA increased eightfold from an EC50 value of 3.39 mM (control) to 0.45 mM, accompanied by a 2.2-fold increase in intrinsic efﬁcacy. Earlier, the authors using the same procedure showed that CGP7930 also affected the dual modulation by a sixfold increase in GABA potency from an EC50 value of 4.9 mM (control) to 0.8 mM and a 1.4-fold increase in efﬁcacy (38). Allosteric Modulators of mGluRs There is a great similarity in the overall structures of metabotropic glutamate and GABAB receptors. Both receptors are dimers with a large N-terminal containing a bilobal structured VFT, seven transmembrane helices, and a large carboxyl terminal. However, the mGluRs are homodimers (41), whereas the GABAB receptor is a heterodimer (see Section ‘‘Allosteric Modulators of GABAB Receptors’’). The positive, negative, and neutral effects of benzaldazine derivatives (Fig. 8) on mGlu5 receptors were described by O’Brien et al. (42). Shifting the ﬂuoro atoms in 3,30 -diﬂuorobenzaldazine (DFB) to the 2,20 position did not change the positive allosteric activity, but there was a 6.5-fold loss in potency. A shift to the 4,40 position changed the allosteric activity from positive to negative with a great (approximately 40-fold) loss in potency. Substituting the 3,30 -diﬂuoro atoms in DFB for 3,30 -dimethoxy groups yielded a negative allosteric compound (3,30 -methoxybenzaldazine, DMeOB) with a potency similar to that of DFB. Replacing the 3,30 -diﬂuoro atoms in DFB by 3,30 -dichloro atoms yielded a nonallosteric neutral compound (3,30 -dichlorobenzaldazine, DCB). A ﬂuorimetric assay of the Ca2þ response was used in evaluating the potentiation by DFB of glutamate (300 nM)

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Figure 8 Allosteric modulators of metabotropic glutamate (mGlu) receptors.

activating human mGlu5 receptors expressed in CHO cells. Maximal potentiation was threefold and the EC50 value was 2.6 mM. In the ﬂuorimetric assay DMeOB in the presence of an EC10 concentration of glutamate acted as a noncompetitive inhibitor with an IC50 value of 3 mM. In the presence of a ﬁxed concentration of DMeOB the concentration–effect curve of glutamate in this assay was shifted to the right. The maximal effect was also reduced, indicating a noncompetitive behavior of negative modulation. DCB inhibits the activity of DFB, DmeOB, and other modulators such as 2-methyl-6-(phenylethynyl)-pyridine, but not the mGlu receptor agonists glutamate or quisqualate. Schaffhauser et al. (43) reported the characteristics of LY 487379 (Fig. 8), a selective and positive allosteric modulator of mGlu2 receptors. The mGlu2 receptor agonists glutamate, (2S,20 R,30 R)-2-dicarboxycyclopropylglycine (DCG-IV), and (2S,1 0 S,2 0 S)-2-carboxycyclopropylglycine (LCCG-1) stimulated [35S]GTPcS binding to a recombinant human mGlu2 receptor preparation in the absence or presence of 3 mM of LY 487378. LY 487379 enhanced potency (EC50) and efﬁcacy (Emax) for glutamate 2.4-fold and 1.1-fold, for DCG-IV 1.5-fold and 2.3-fold, and for LCCG-1 5.3fold and twofold, respectively. Mutagenic analysis showed that amino acids Ser688 (TM4), Gly689 (TM4), and Asn735 (TM5) of the mGlu2 receptor are important for binding of the positive allosteric modulator LY 487379. Allosteric Modulators of Calcium-Sensing (Ca2þ) Receptors Allosteric enhancers of Ca2þ receptors (calcimimetics) have been known since 1998 (44). The best studied compound is NPS R-568 (Fig. 9). By acting selectively on Ca2þ receptors of the parathyroid gland, NPS R-568 inhibits the secretion of parathyroid hormone (PTH) in vitro (bovine parathyroid cells, IC50 ¼ 27 nM) and in vivo (rat, ED50 ¼ 1.1 mg/kg p.o.) (44). In patients with primary or secondary hyperparathyroidism the PTH secretion was suppressed by NPS R-568. Unfortunately, the pharmacodynamic and pharmacokinetic data in these patients showed a high variability. Further

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Figure 9 Allosteric modulators of calcium-sensing receptors.

development of NPS R-568 has been stopped [for review see Fraza˜o (45)]. Cinacalcet (AMG 073) (Fig. 9), lacking the metabolically labile methoxy group, has a better pharmacokinetic proﬁle than NPS R-568. Cinacalcet also inhibits the PTH secretion in bovine parathyroid cells (IC50 ¼ 27 nM) and causes a dose-dependent long-lasting reduction in serum PTH levels in rats (8 hours at 10 mg/kg p.o.) (46). Cinacalcet is currently undergoing phase III clinical trials (see Section ‘‘Clinical Studies’’). A novel calcimimetic is calindol (Fig. 9); it is equipotent to NPS R-568 in increasing the Ca2-induced accumulation of inositol phosphates in HEK293 cells expressing the human Ca2þ receptor (EC50 values of 0.31 and 0.50 mM, respectively) (47). The ﬁrst selective allosteric antagonist of Ca2þ receptors (calcilytic) is NPS 2143 (Fig. 9) [for review see (48)]. The compound stimulates PTH secretion from bovine parathyroid cells (EC50 ¼ 41 nM) and enhances the plasma PTH levels in normal rats after intravenous infusion (0.1 mmol/kg/min).

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Since intermittently injected PTH in osteoporotic patients has anabolic activity on bone, the effects of NPS 2143 were assessed in ovariectomized rats, an animal model of osteoporosis. Daily oral administration of both NPS 2143 (100 mmol/kg) and 17-b-estradiol for ﬁve weeks resulted in net bone gain. Coadministration of 17-b-estradiol is necessary to prevent bone resorption induced by the prolonged elevated plasma PTH level. Shorter-acting calcilytics, intermittently administered, will induce transient increases in plasma PTH levels, mimicking the physiology of PTH release much better. This goal was achieved by replacing the 2-naphthyl moiety of NPS 2143 by a 4-methoxyphenyl group. Using the (R)-enantiomers and appropriate substituents on the phenoxy group reduced the potential b-adrenoceptor blocking activity. A calcilytic (structure not disclosed) entered phase 1 clinical trials for the treatment of osteoporosis (NPS Pharmaceuticals Inc., Salt Lake City, Utah, U.S.A.). A novel calcilytic is Calhex 231 (Fig. 9). It blocks the Ca2þ-induced accumulation of inositol phosphates in HEK293 cells transiently expressing the human Ca2þ receptor (47). The compound is equipotent to NPS 2143 (IC50 values of 0.39 and 0.35 mM, respectively). Mutagenesis studies in HEK293 cells transiently expressing the human wild-type Ca2þ receptors and a number of mutants showed that the binding pockets of calcimimetics (NPS 568 and calindol) and calcilytics (NPS 2143 and Calhex 231) are partially overlapping but not identical (47). The effects of these compounds on the Ca2þ-induced production of inositol phosphates are absent in the Glu837Ala mutant. These results show a crucial role of this negatively charged glutamate in TM7 in anchoring the compounds via an H-bridge with the protonated secondary amine of the calcimimetics and calcilytics. The lack of potentiation of the Ca2þ-induced production of inositol phosphates by NPS 568 and calindol in the Ile841Ala mutant (TM7) indicates the involvement of Ile841 in the binding of both calcimimetics. However, the compounds interact with different amino acids on TM6. NPS 568 does so with Phe821 and calindol with Trp818. The calcilytic NPS 2143 lost its ability to inhibit the Ca2þ-induced accumulation in the mutants Phe684Ala and Phe688Ala (both TM3). These results show that Phe684 and Phe688 participate in NPS 2143 recognition. A 12-fold decrease in the effect of NPS 2143 was observed after mutation of Arg680 on TM3 and Ile841 on TM7. It is obvious that Arg680, located next to the crucial Phe684 and Phe688, and Ile841, located next to the crucial Glu837, are also involved in the binding of NPS 2143. Similar to NPS 2143, Calhex 231 interacts with Phe684 located on TM3 and with Glu837 on TM7. However, in contrast to NPS 2143, the mutation Phe688Ala is not detrimental to Calhex 231 binding. The effect of Calhex 231 is about eight times less than that observed for the wild-type Ca2þ receptor. Moreover, the mutation Arg680Ala on TM3 is favorable for Calhex 231 binding (a threefold increase in potency). Other favorable mutations are Leu776Ala on TM5 and Phe821Ala on TM6 (an 18-fold increase in potency). These mutants allow better accommodation of Calhex 231 in the ligand

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binding pocket of the Ca2þ receptor. Favorable mutants for NPS 2143 were not observed. CLINICAL STUDIES With the emerging interest in allosteric modulation it does not come as a surprise that some chemical entities have now progressed from the preclinical to clinical phases of investigation. With respect to class A receptors the allosteric enhancer of adenosine A1 receptors T62 (Fig. 2), potentially useful in the treatment of neuropathic pain [see also (17)], has been tested on a limited number of healthy volunteers to allow for dose ﬁnding. The results of this trial have not been made available yet (18). One particular CRF1 receptor (class B) NPA, R-121919 (Fig. 5), entered the phase IIa studies. These have been discontinued, although the compound ameliorated depressive symptoms without undesired endocrine effects (33). The calcium-sensing agonist cinacalcet (Fig. 9) underwent various phase III clinical trials for the treatment of secondary hyperparathyroidism in hemodialysis patients (49,50). The results of the most extended study so far have been reported recently (50). Over 700 patients were involved in two identical randomized, double-blind, placebo-controlled trials. The patients received cinacalcet (30–180 mg/day, p.o., n ¼ 371) or placebo (n ¼ 370) for 26 weeks. The treatment phases consisted of a 12-week dose-titration period followed by a 14-week efﬁcacy assessment. The 26 weeks of treatment were completed by 68% of the patients on cinacalcet and 78% of the placebo group. Cinacalcet decreased the plasma PTH levels in a dose-dependent way. Mean PTH levels decreased by 30% in 64% of patients given cinacalcet. Treatment with cinacalcet was associated with moderate reductions in serum calcium (6.8%) and phosphate (8.4%) levels, and was well tolerated. The gastrointestinal side effects were generally mild to moderate and transient. CONCLUDING REMARKS Can all GPCRs be modulated allosterically? The answer to this question may indeed by afﬁrmative. The speciﬁc examples for all three GPCR classes (1, 2, and 3) discussed above suggest that this is the case. This hypothesis could then be the more general starting point for a search for new chemical entities that are modulators of GPCR activity, unlike directly acting (orthosteric) ligands such as agonists and antagonists. Most screening assays, particularly those in high-throughput format, are, however, not necessarily organized such that the dissociation and association kinetics of the receptor–ligand interaction are also taken into account. Rather than a two-component screening, i.e., an assay with (labeled) receptor preparation and compound, a three-component system with a potential allosteric

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modulator present should then be considered. This experimental setup may well be feasible and could be used in both radioligand binding studies and assays with ﬂuorescent probes, either addressing interaction with the receptor itself or through postreceptor signaling events. It is unfortunate that appropriate experimental evidence is often lacking, when ligands are claimed to be allosteric in nature. Only rarely are the effects on receptor–ligand dissociation kinetics studied, to name just one shortcoming. The latter remark stems from the classical deﬁnition of an allosteric modulator as a compound that does not have receptor activity of its own. It can only be active in the presence of an (orthosteric) agonist or antagonist. It functions by inﬂuencing the dissociation from or association to the receptor of the agonist or antagonist (please note though that the association kinetics are also inﬂuenced by competitive—orthosteric— ligands). Within this strict framework it is obvious that many ligands mentioned in this review do not truly qualify as allosteric modulators. AC-42 and N-desmethylclozapine (Fig. 4) are typical examples of ligands that each independently activate the muscarinic M1 receptor via sites that are distant from the acetylcholine binding site. Also, for the class B and C receptors it seems that the allosteric modulators can be very active indeed in the absence of the endogenous agonist. Most striking in this respect is the observation that the mGlu5 receptor with a deleted VFT containing the N-terminal domain (the glutamate binding site) can be fully activated by 3,30 -DFB (Fig. 8) (51). Can these observations be rationalized? In Figure 10, cartoons of GPCRs from classes 1–3 are graphically represented. For most known class 1 receptors the endogenous ligand (neurotransmitter or hormone) is believed to bind and activate the receptor from within the cavity formed by the seven TM domains. There might be little room for orthosteric and allosteric ligands to have fully independent binding sites. The situation may be different for the class 2 and 3 receptors, where the endogenous ligands generally bind to the N-terminal domains, leaving the TM cavity essentially unoccupied with substantial room for other (allosteric) ligands to bind and even activate the receptor. The last important issue is the deﬁnition of an allosteric modulator. If we maintain the strict description in which the kinetics of the receptor– ligand interaction are very important, we propose to use the term ‘‘noncompetitive agonism’’ or ‘‘allosteric agonism’’ for compounds such as AC-42, N-desmethylclozapine, and DFB. This might remove some of the confusion, and, more importantly, provide a rational dichotomy for the classiﬁcation of novel compounds. In conclusion, owing to the wide tissue distribution of GPCRs, directly acting (endogenous) agonists or antagonists often exhibit side effects associated with the activation of receptors in tissues other than the therapeutic target. Allosteric modulators, in contrast, may induce tissue and receptor

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Figure 10 Schematic representations of G-protein–coupled receptors (GPCRs) from the three classes 1, 2, and 3 (see also text). The putative orthosteric binding site may (class 1) or may not (class 2 and 3) be close to the allosteric binding site.

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subtype selectivity. They enhance or diminish the effects of (endogenous) agonists that are often conﬁned to or produced in the afﬂicted tissue only. Hence, allosteric modulators may in fact be a potentially safer category of drugs. The recent surge in clinical trials with allosteric modulators may be a reﬂection of this assumption. REFERENCES 1. Drews J. Drug discovery: a historical perspective. Science 2000; 287:1960–1964. 2. Monod J, Changeux JP, Jacob F. Allosteric proteins and cellular control systems. J Mol Biol 1963; 6:306–329. 3. Bockaert J, Pin JP. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J 1999; 18:1723–1729 (see also the public-domain receptor database at www.gpcr.org). 4. Soudijn W, Van Wijngaarden I, IJzerman AP. Allosteric modulation of G protein-coupled receptors. Perspectives and recent developments. Drug Discov Today 2004; 9:752–758. 5. Christopoulos A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nature Rev Drug Discov 2002; 1:198–210. 6. Christopoulos A, Kenakin T. G protein-coupled receptor allosterism and complexing. Pharmacol Rev 2002; 54:323–374. 7. Bruns RF, Fergus JH. Allosteric enhancement of adenosine A1 receptor binding and function by 2-amino-3-benzoylthiophenes. Mol Pharmacol 1990; 38: 939–949. 8. Bruns RF, Fergus JH, Coughenour LL, et al. Structure–activity relationships for enhancement of adenosine A1 receptor binding by 2-amino-3-benzoylthiophenes. Mol Pharmacol 1990; 38:950–958. 9. Bhattacharya S, Linden J. The allosteric enhancer, PD 81,1723, stabilizes human A1 adenosine receptor coupling to G proteins. Biochem Biophys Acta Mol Cell Res 1995; 1265:15–21. 10. Mizumura T, Auchampach A, Linden J, Bruns RF, Gross GJ. PD81,723, an allosteric enhancer of the A1 adenosine receptor, lowers the threshold for ischemic preconditioning in dogs. Circ Res 1996; 79:415–423. 11. Battacharya S, Linden J. Effects of long-term treatment with the allosteric enhancer, PD 81,723, on Chinese hamster ovary cells expressing recombinant human A1 adenosine receptors. Mol Pharmacol 1996; 50:104–111. 12. Shryock JC, Ozeck MJ, Belardinelli L. Inverse agonists and neutral antagonists of recombinant human A1 adenosine receptors stably expressed in Chinese hamster ovary cells. Mol Pharmacol 1998; 53:886–893. 13. Van der Klein PA, Kourounakis AP, IJzerman AP. Allosteric modulation of the adenosine A1 receptor. Synthesis and biological evaluation of novel 2-amino-3-benzoylthiophenes as allosteric enhancers of agonist binding. J Med Chem 1999; 42:3629–3636. 14. Tranberg CE, Zickgraf A, Ciunta BN, et al. 2-Amino-3-aroyl-4,5-alkylthiophenes: agonist allosteric enhancers at human A1 adenosine receptors. J Med Chem 2002; 45:382–389.

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31. Hoare SR, Sullivan SK, Ling N, Crowe PD, Grigoriadis DE. Mechanism of corticotropin-releasing factor type I receptor regulation by nonpeptide antagonists. Mol Pharmacol 2003; 63:751–765. 32. Hoare SR, Sullivan SK, Schwarz DA, et al. Ligand afﬁnity for amino-terminal and juxtamembrane domains of the corticotropin releasing factor type I receptor: regulation by G-protein and nonpeptide antagonists. Biochemistry 2004; 43:3996–4011. 33. Holsboer F. Corticotropin-releasing hormone modulators and depression. Curr Opin Investig Drugs 2003; 4:46–50. 34. Madsen P, Ling A, Plewe M, et al. Optimization of alkylidene hydrazide based human glucagon receptor antagonists. Discovery of the highly potent and orally available 3-cyano-4-hydroxybenzoic acid [1-(2,3,5,6-tetramethylbenzyl)-1Hindol-4-ylmethylene]hydrazide. J Med Chem 2002; 45:5755–5775. 35. Runge S, Gram C, Brauner-Osborne H, Madsen K, Knudsen LB, Wulff BS. Three distinct epitopes on the extracellular face of the glucagon receptor determine speciﬁcity for the glucagon amino terminus. J Biol Chem 2003; 278:28005–28010. 36. Tibaduiza EC, Chen C, Beinborn M. A small molecule ligand of the glucagonlike peptide 1 receptor targets its amino-terminal hormone binding domain. J Biol Chem 2001; 276:37787–37793. 37. Kniazeff J, Galvez T, Labesse G, Pin JP. No ligand binding in the GB2 subunit of the GABAB receptor is required for activation and allosteric interaction between the subunits. J Neurosci 2002; 22:7352–7361. 38. Urwyler S, Mosbacher J, Lingenhoehl K, et al. Positive allosteric modulation of native and recombinant c-aminobutyric acidB receptors by 2,6-Di-tert-butyl-4(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and its aldehyde analog CGP13501. Mol Pharmacol 2001; 60:963–971. 39. Onali P, Mascia FM, Olianas MC. Positive regulation of GABAB receptors dually coupled to cyclic AMP by the allosteric agent CGP7930. Eur J Pharmacol 2003; 471:77–84. 40. Urwyler S, Pozza MF, Lingenhoehl K, et al. N,N’-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4, 6-diamine (GS39783) and structurally related compounds: novel allosteric enhancers of c-aminobutyric acidB receptor function. J Pharmacol Exp Ther 2003; 307:322–330. 41. Kunishima N, Shimada Y, Tsuji Y, et al. Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 2000; 407:971–977. 42. O’Brien JA, Lemaire W, Chen TB, et al. A family of highly selective allosteric modulators of the metabotropic glutamate receptor subtype 5. Mol Pharmacol 2003; 64:731–740. 43. Schaffhauser H, Rowe BA, Morales S, et al. Pharmacological characterization and identiﬁcation of amino acids involved in the positive modulation of metabotropic glutamate receptor subtype 2. Mol Pharmacol 2003; 64:798–810. 44. Nemeth EF, Steffey ME, Hammerland LG, et al. Calcimimetics with potent and selective activity on the parathyroid calcium receptor. Proc Natl Acad Sci USA 1998; 95:4040–4045. 45. Fraza˜o JM, Martins P, Coburn JW. The calcimimetic agents: perspectives for treatment. Kidney Int Suppl 2002; 61:S149–S154.

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46. Nemeth EF, Heaton WH, Miller M, et al. Pharmacodynamics of the type II calcimimetic compound cinacalcet HCl. J Pharmacol Exp Ther 2004; 308:627–635. 47. Petrel C, Kessler A, Dauban P, Dodd RH, Rognan D, Ruat M. Positive and negative allosteric modulators of the Ca2þ-sensing receptor interact within overlapping but not identical binding sites in the transmembrane domain. J Biol Chem 2004; 279:18990–18997. 48. Nemeth EF. The search for calcium receptor antagonists (calcilytics). J Mol Endocrinol 2002; 29:15–21. 49. Goodman WG, Hladik GA, Turner SA, et al. The calcimimetic agent AMG073 lowers plasma parathyroid hormone levels in hemodialysis patients with secondary hyperparathyroidism. J Am Soc Nephrol 2002; 13:1017–1024. 50. Block GA, Martin KJ, De Francisco ALM, et al. Cinacalcet for secondary hyperparathyroidism in patients receiving hemodialysis. N Engl J Med 2004; 350:1516–1525. 51. Goudet C, Gaven F, Kniazeff J, et al. Heptahelical domain of metabotropic glutamate receptor 5 behaves like rhodopsin-like receptors. Proc Natl Acad Sci USA 2003; 101:378–383.

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9 Allosteric Modulators of Group III Metabotropic Glutamate Receptors as Novel Therapeutics Jesper Mosolff Mathiesen Department of Molecular Pharmacology, H. Lundbeck A/S, Valby and Department of Medicinal Chemistry, The Danish University of Pharmaceutical Sciences, Copenhagen, Denmark

M. Teresa Ramirez Department of Molecular Pharmacology, Zealand Pharma A/S, Glostrup, Denmark

METABOTROPIC GLUTAMATE RECEPTORS IN GLUTAMATERGIC NEUROTRANSMISSION Glutamate is the major and most abundant excitatory neurotransmitter in the central nervous system (CNS). Due to its presence in most species, this amino acid represents one of the oldest neurotransmitters in nature. The regulation and activity of glutamate in the synapse is orchestrated by no less than three families of ionotropic receptors, three groups of metabotropic receptors, and ﬁve transporters (1). The existence of multiple subtypes and splice variants within each of these families and groups further adds to the complexity of this system. Contributing to the intricacy of glutamatergic regulation is the presence of allosteric sites for modulators in addition to the orthosteric glutamate binding site. The discovery of allosteric binding sites within the seven transmembrane (7TM) domains of the metabotropic glutamate receptors (mGluRs)

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represents a novel mechanism for ﬁne-tuning the effects of glutamate and provides insight into the generation of therapeutics with novel modes of action. Metabotropic Glutamate Receptors Classification The mGluRs are members of the G-protein–coupled family of receptors (GPCRs) and have traditionally been divided into three groups based on their sequence homology, ligand pharmacology, and G-protein–coupling properties (Table 1) (2). Group I consists of subtypes 1 and 5 that are selectively activated by 3,5-dihydroxyphenylglycine (DHPG) and L-quisqualate. Upon activation, Group I mGluRs couple to Gaq, which subsequently activates phospholipase C (PLC) leading to the generation of diacylglycerol and inositol triphosphate. Group II consists of subtypes 2 and 3, which are selectively activated by 1S,2S,5R,6S-2-aminobicyclo (3,1,0) hexane-2,6bicarboxylate monohydrate (LY354740) and 2S,20 R,30 R-2-(20 ,30 -dicarboxycyclopropyl) glycine (DCG-IV), whereas Group III mGluRs (subtypes 4, 6, 7, and 8) are selectively activated by L-(þ)-2-amino-4-phosphonobutyric acid (L-AP4), L-serine-O-phosphate (L-SOP), and R,S-4-phosphonophenylglycine (PPG). Both Group II and III mGluRs couple to Gao/i leading to inhibition of adenylyl cyclase (AC) and a subsequent decrease in cyclic adenosine monophosphate (cAMP) production in heterologous expression systems. In native systems, mGluRs have been shown to activate a number of other effector proteins in addition to PLC and AC (2). Table 1 Classiﬁcation of the mGluR Subtypes

Group I Group II Group III

mGluR1 mGluR5 mGluR2 mGluR3 mGluR4 mGluR6 mGluR7 mGluR8

G-protein coupling

Second messengers

Group selective orthosteric agonists

Gaq

"IP3,"DAG

L-quisqualate,

Gai/o

#cAMP

LY354740, DCG-IV

Gai/o

#cAMP

L-AP4, L-SOP,

DHPG

PPG

Note: These receptors are grouped based on their G-protein–coupling properties, agonist selectivity, and sequence homology (not shown). Abbreviations: mGluR, metabotropic glutamate receptor; DHPG, 3,5-dihydroxyphenylglycine; cAMP, cyclic adenosine monophosphate; L-AP4, L-(þ)-2-amino-4-phosphonobutyric acid; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; LY354740, 1S,2S,5R,6S-2-aminobicyclo (3,1,0) hexane-2-6-bicaroxylate monohydrate; DCG-IV, 2S,20 R,30 R-2-(20 ,30 -dicarboxycyclopropyl) glycine; L-SOP, L-serine-O-phosphate; PPG, R,S-4-phosphonophenylglycine.

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Structure The ligand-binding domain of the mGluRs is unique and characteristic of Family C GPCRs, which include the Ca2þ-sensing, c-aminobutyric acid (GABAB), and pheromone receptors (3). mGluRs are expressed as homodimers and have a large amino-terminal domain (ATD) of approximately 500 amino acids that resembles a Venus ﬂy trap (Fig. 1). Crystallization studies of the mGluR1 ATD have demonstrated that dimerization takes place between the two ATDs of the homodimer by noncovalent interactions that are further stabilized by an intermolecular disulﬁde bond (4,5). The endogenous ligand L-glutamate binds to the homodimer in a binding pocket within the ATD. The glutamate binding site is highly conserved among the mGluRs and represents the orthosteric site. Closure of one of the ATDs in the receptor dimer results in a conformational change of the two ATDs relative to each other, which is transposed to the 7TM domain and leads to receptor activation. Recent studies have suggested that the two 7TM domains of the dimer rearrange upon ligand binding in the ATD to form an active 7TM domain conformation (6). Binding of L-glutamate to the binding pocket increases the probability of the ATD existing in its closed state. It has been proposed that closure of one ATD only activates the receptor partially, while closure of both ATDs is required for full activation (7). Over the last decade, a number of compounds that all lack the a-aminoacidic moiety characteristic of competitive ligands and that do not displace

Figure 1 Sketch of the mGluR homodimer. The large ATD contains the L-glutamate-binding pocket. The dimer is interconnected by noncovalent forces and a disulﬁde bridge located in the ATD. A Cys-rich domain is located between ATD and the seven transmembrane domains (7TM domain); the latter characteristic of all GPCRs. Abbreviations: mGluR, metabotropic glutamate receptor; ATD, aminoterminal domain; Cys-rich, cystein-rich; GPCRs, G-protein–coupled receptors.

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orthosteric ligands have been identiﬁed for mGluRs. These compounds act as either positive or negative allosteric modulators and bind in the 7TM domain where they exert their modulatory effects. Allosteric modulators, models, and mechanisms will be discussed later in detail. Presynaptic mGluR Localization and Function Presynaptic mGluRs are key regulators of glutamate release from the presynaptic neuron. They are believed to serve as autoreceptors that sense the glutamate concentration present in the synaptic/extrasynaptical space and subsequently terminate the release of glutamate from the presynaptic neuron. Presynaptic mGluRs belong primarily to Group II and III [reviewed by Schoepp (8)], although there is also electrophysiological evidence for a presynaptic Group I mGluR (9). Besides coupling to Gao/i and the inhibition of AC, a number of in vivo studies suggest that Group II and III mGluRs couple to other signaling systems. For example, inhibition of presynaptic Ca2þ channels by Gao/idependent and independent mechanisms have been shown for Groups II and III with regard to their autoregulatory function (10–13). Furthermore, Group III mGluR activation has been shown to decrease synaptic vesicle exocytosis as well as to modulate vesicle recovery and endocytosis in aortic baroreceptor neurons (14). Similar mechanisms are likely to exist for Group III autoreceptors in other parts of the CNS. In addition other non Ca2þ-channel–mediated mechanisms of autoreceptor-induced inhibition of glutamate release have been proposed (15). The localization of the various Group II and III mGluR subtypes in the CNS is highly dependent on the speciﬁc regions studied [for a detailed description see Schoepp (8)]. Most of the information regarding the subcellular localization of Groups II and III on the single-cell level comes from hippocampal and cerebellular immunocytochemical studies, although these receptors are widely distributed in most of the CNS. mGluR2 is primarily located outside the active zone on preterminal neuronal elements, whereas mGluR3 is mainly localized to glial cells. Both subtypes are proposed to be sensors of glutamate diffusion from the synapse into the extrasynaptical space. Group III mGluRs are predominantly located in the vicinity of the presynaptic active zones in both glutamatergic and nonglutamatergic synapses. This localization pattern suggests an autoreceptor function at glutamatergic synapses as well as a presynaptic heteroreceptor one, i.e., the regulation of neurotransmitter release besides that of glutamate such as GABA in GABAergic synapses (16). In support of a role for the mGluRs as glutamatergic autoreceptors, there is a strong correlation between their proximity to the active zone of glutamate release and their sensitivity towards glutamate (Fig. 2). The only Group III mGluR expressed inside the active zone, mGluR7, has low

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Figure 2 Localization of the various mGluR subtypes in the glutamatergic synapse. The numbers indicate the mGluR subtype and the small circles represent glutamate molecules. The localization of the three ionotropic glutamate receptors in the postsynaptic active zone (KAIN, AMPA, and NMDA) is also indicated. Abbreviations: mGluR, metabotropic glutamate receptor; KAIN, kainate; AMPA, 2-amino3-hydroxy-5-methylisoxazole-4-propionic acid; NMDA, N-methyl-D-aspartate.

millimolar afﬁnity towards glutamate, which is the glutamate concentration range reached upon presynaptic release of glutamate. mGluR4 and mGluR8 display low micromolar afﬁnities toward glutamate consistent with their peri/extrasynaptic localizations on the presynaptic neuron further away from the active zone. Group II mGluRs are likely to serve as sensors for glutamate spillover or diffusion from the active zone of the synaptic cleft. It should be noted that the exact localization and composition of the mGluR subtypes in neurons are highly dependent on the brain region studied and even vary between different terminals on the same axon. This suggests quite specialized roles in neurotransmission for each of the Group III mGluRs. Since only a few of the currently known ligands for Group III mGluRs display some selectivity toward a single subtype, the role of the individual subtypes in autoregulation is unclear. Postsynaptic mGluR Localization and Function Once released into the synaptic cleft, glutamate activates postsynaptic glutamate receptors that mediate excitatory neurotransmission. The fast excitatory response in the postsynaptic neuron is mediated primarily by the ionotropic glutamate receptors (iGluRs). The iGluRs are cationic channels composed of four subunits that together form a pore in the membrane. iGluRs have been divided into three families based on their selective agonists: 2-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA). Within each of these families, several subunits have been identiﬁed and characterized according to their

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subunit compositions and their conductances for sodium and calcium ions. The iGluRs are mainly concentrated in the postsynaptic density facing the glutamate release site (Fig. 2) (17). The mGluRs belonging to Group I are also located postsynaptically. Immunocytochemical studies have demonstrated that postsynaptic mGluRs are located adjacent to the synaptic cleft or extrasynaptically, reﬂecting their function as modulators of fast excitatory neurotransmission (17). Group I mGluRs are believed to serve as both immediate and delayed regulators of postsynaptic excitability through their immediate or downstream effectors. For example, activation of Group I mGluRs has been shown to increase excitability of neurons by potentiating the NMDA receptor response (18,19), while the modulation of Kþ and Ca2þ channels by Group I mGluRs also affects the overall neuronal excitability [reviewed by Conn and Pin (2)]. ROLE OF GROUP III mGluRs IN CNS DISORDERS Because of their broad distribution in the CNS and their role in glutamatergic neurotransmission, mGluRs play an important role not only in CNS functions such as learning and motor control but also in pathological conditions. Group I inhibition and Groups II and III activation, for instance, may have potential beneﬁts in the treatment of CNS disorders where glutamatergic hyperactivity is pathophysiological. Additionally, these opposing activities are neuroprotective in a number of in vitro and in vivo models of chronic neurodegeneration and acute neurotoxic traumas (20). Since mGluRs also modulate the release of a vast number of other neurotransmitters such as excitatory and inhibitory amino acids, monoamines, and neuropeptides, the effects of mGluR modulation are indeed complex (16). Epilepsy A string of studies suggest that mGluRs are involved in epileptogenesis and seizure discharge. The general role of mGluRs in epilepsy has been demonstrated by the proconvulsant effect of Group I activation and the anticonvulsant effects of Groups II and III activation in models of epilepsy. For instance, Group III mGluR agonists L-SOP, L-AP4 (RS)-1-amino-3(phosphonomethylene)-cyclobutane-1-carboxylic acid (cyclobutylene AP5), and PPG block seizures in several rodent models of complex partial and absence seizures (21). The involvement of Group III receptors in epilepsy has been further substantiated in knockout mice and receptor expression studies as well as the effect of seizure kindling on synaptic transmission. mGluR4 knockout (/) mice showed resistance to GABAA antagonist-induced absence seizures (22) and mGluR7 knockout (/) mice developed epilepsy after 12 weeks (23). In support of Group III involvement in epilepsy, upregulation of mGluR4 and mGluR7 in the hippocampus has been observed in human

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temporal lobe epilepsy and kindled rat models (24,25). In kindling studies, the effect of mGluR4 and possibly mGluR8 activation on synaptic transmission is either impaired or enhanced compared to control animals depending on the part of the brain studied [reviewed by Moldrich et al. (21)]. Together, these studies suggest that seizure vulnerability is related to the function and expression of Group III mGluRs and that upregulation of Group III most likely serves as an autoregulatory mechanism to counteract the enhanced excitatory activity observed in epilepsy. Parkinson’s Disease A link between mGluR4 and Parkinson’s disease has recently been established, where both mGluR4 orthosteric and allosteric activation have been shown to have antiparkinsonian effects. In acute and chronic models of Parkinson’s disease, L-AP4 decreased transmission at the striatopallidal synapse through its function as a heteroautoreceptor of GABAergic neurotransmission (26). The effect of L-AP4 was absent in mGluR4 knockout mice. The mGluR4 allosteric modulator N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide (PHCCC) (discussed in detail in section ‘‘Group III mGluR allosteric modulators’’) also had an antiparkinsonian effect in a dopamine depletion akinesia model, which again was attributed to the function of mGluR4 as a heteroautoreceptor of GABAergic neurotransmission (27). Anxiety and Depression Glutamatergic neurotramission has also been associated with anxiety and depression. Activation of Group III mGluRs by agonists and speciﬁcally mGluR4 by PHCCC shows anxiolytic-like effects in the conﬂict-drinking test. Group III orthosteric agonists also exhibited antidepressant effects (28–30). Neurodegeneration Substantial degrees of neurodegeneration are observed in a number of diseases including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and in the neurodegenerative element and progression of epilepsy. Group III activation is neuroprotective in a number of in vitro and in vivo models. The neuroprotective effect of Group III activation has primarily been associated with the inhibition of glutamate release. However, other neuroprotective effects of Group III activation have been proposed, e.g., activation of the mitogen-activated protein kinase and PI-3-K pathways in cerebellar granule cells undergoing apoptosis due to potassium deprival (31). A neuroprotective effect of the mGluR4-positive allosteric modulator ()-PHCCC in NMDA-induced excitotoxicity and b-amyloid-peptide–induced neurotoxicity in mixed cortical neurons was recently described. ()-PHCCC had a neuroprotective effect alone, most likely due to the presence of ambient glutamate,

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and enhanced the effect of orthosteric agonist-mediated neuroprotection (32). In support for the importance of mGluR4 expression in neuroprotection, a decrease in mGluR4 mRNA levels has been associated with apoptosis of potassium-deprived cerebellar granule cells, whereas culture survival increases when mGluR4 expression is elevated or Group III receptors are activated (33). Neuroinflammation Another aspect of neuronal death is neuroinﬂammation. In addition to their role of mediating responses that limit tissue damage, inﬂammatory mediators are neurotoxic under certain conditions (34). Recently, activation of Group III mGluRs has been shown to inhibit the release of neurotoxic glutamate from microglia overstimulated by chromogranin A, lipopolysaccharide, and b-amyloid-peptide25–35 (35). Furthermore, the subsequent neurotoxic effects on neurons induced by chromogranin A and lipopolysaccharide were attenuated by Group III activation. Activation of Group III mGluR by L-AP4 has also been shown to inhibit the synthesis and release of the chemokine RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) in astrocytes activated by proinﬂammatory cytokines and to improve functional recovery in an in vivo model of neuroinﬂammation (36). Since the effects of L-AP4 correspond to mGluR4 pharmacology and since the effect of L-AP4 in mGluR4 (/) knockout mice was impaired, inhibition of RANTES release appeared to be primarily mediated by mGluR4. Together with Western blot analysis that conﬁrmed the expression of mGluR4 in murine astrocytes, the data strongly indicate that mGluR4 is responsible for the inhibition of RANTES release from cytokine-activated astrocytes. Thus, activation of Group III mGluRs may have beneﬁcial effects in neuroinﬂammatoryassociated diseases such as sclerosis, Alzheimer’s disease, and stroke. MODELS OF mGluR ALLOSTERIC MODULATION With the identiﬁcation of allosteric modulators for GPCRs, a need has arisen for models that describe the relationship between allosteric modulators and their effects on orthosteric ligand activation. Previous models of GPCR allosteric modulation have focused on the interaction of the G-protein with the receptor where the G-protein is deﬁned as the modulator (37). In the case of Family A GPCRs where both the orthosteric and allosteric ligand binding sites are located in the 7TM domain, close cooperativity between these sites is expected. Previously well-described models used for understanding receptor– G-protein interactions have been employed to understand allosteric interactions between a GPCR and its orthosteric and allosteric ligands (38). However, can these models be used to explain allosteric modulation in the Family C GPCRs where the orthosteric site is extracellular and the

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allosteric site is located within the 7TM domain? Adding to this complexity are the facts that this family of receptors exists as homodimers and that it is unclear whether the 7TM domain from each receptor in the homodimer come together to form an allosteric site distinct from that found in the monomer. Thus, it is conceivable that modulation in the Family C GPCRs may give rise to allosteric effects not previously described by models where close cooperativity between two domains is expected within a single receptor. Positive Modulators in the Allosteric Two-State Model In a model described by Hall (38), a positive allosteric modulator binding to a receptor may stabilize either (i) a receptor conformation with higher afﬁnity for the orthosteric agonist, i.e., ‘‘binding cooperativity,’’ or (ii) a receptor conformation where the orthosteric agonist has higher afﬁnity for the active compared to the inactive receptor conformation, i.e., ‘‘activation cooperativity.’’ A positive allosteric modulator that stabilizes a receptor conformation where the orthosteric agonist has higher afﬁnity for the active receptor conformation as in the latter situation may consequently increase the apparent afﬁnity of the receptor for the orthosteric agonist and thus resemble the positive allosteric modulator described in the former situation in afﬁnity binding studies. A likely consequence of binding cooperativity in a functional assay is an increase in the potency of the orthosteric agonist without an increase in orthosteric ligand efﬁcacy, since the positive allosteric modulator will only increase the afﬁnity of the receptor for the orthosteric agonist. The consequence of activation cooperativity in a functional assay is an increase in both potency and efﬁcacy of the orthosteric ligand. In the presence of the orthosteric agonist, the positive allosteric modulator is expected to increase the afﬁnity of the orthosteric ligand for the active receptor conformation, resulting in an equilibrium shift toward the active conformation. As in (i), the increase of the apparent afﬁnity for the orthosteric agonist is reﬂected as an increase in potency in a functional assay. However, since the ratio of active to inactive receptor is shifted toward the active receptor conformation, the apparent intrinsic efﬁcacy of the orthosteric agonist may also increase, resulting in a higher efﬁcacy of the orthosteric agonist. Finally, the positive allosteric modulator may stabilize the active receptor conformation on its own and thus have intrinsic agonist activity, i.e., allosteric agonism. By stabilizing the active receptor conformation, the allosteric agonist may increase the apparent afﬁnity of the orthosteric agonist. The likely outcome in a functional assay would be an agonistic effect by the allosteric agonist when applied alone. Furthermore, the allosteric agonist would be expected to increase both the potency and the efﬁcacy of an orthosteric agonist since the effect of the allosteric agonist would synergize with the effect of the orthosteric agonist (38).

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A Model for Family C GPCRs In Family A, both the orthosteric and allosteric binding sites are located in the 7TM domain. While a high degree of cooperativity between the orthosteric and allosteric sites in Family A is expected, this may not be the case for Family C where the orthosteric binding site is clearly distinct from the allosteric site. There is experimental evidence suggesting that some allosteric modulators of Family C GPCRs do not affect the apparent afﬁnity of the orthosteric agonist as predicted for an allosteric modulator in Family A (38). By combining a proposed model for ligand binding to bacterial periplasmic-binding proteins, which are believed to be the ancestor to the mGluR ATD, with a two-state model of GPCRs, the model of Parmentier (39) can explain the experimental observation that allosteric modulators do not affect the afﬁnity of the orthosteric agonist. This is based on the notion that a conformational change in the 7TM domain does not necessarily cause a conformational change in the orthosteric-binding domain and vice versa. Instead, the functional coupling between the two domains in Family C may be either ‘‘tight’’ or ‘‘loose.’’ If the coupling is ‘‘tight,’’ a Family C GPCR will probably behave like a Family A GPCR where there is a high degree of cooperativity between the orthosteric and allosteric binding domains and where a positive allosteric modulator increases the orthosteric agonist afﬁnity. Conversely, if the coupling is ‘‘loose,’’ the degree of cooperativity is low and the binding of a positive allosteric modulator will most likely not increase the orthosteric agonist afﬁnity. Thus, the effect of an allosteric modulator on a Family C GPCR is dependent on the coupling properties between the ATD and 7TM domains of the receptor. In the model proposed by Parmentier (39), an allosteric modulator may either change the equilibrium between the active and inactive states of the 7TM domain or modify the coupling between the 7TM and the ligand-binding domains. Moreover, it is possible to explain why some orthosteric and allosteric ligands affect the basal/constitutive activity of a receptor while others do not (39). We will revisit the models proposed by Hall and Parmentier in relation to the observed effects of the allosteric modulators described in the following sections. It should be noted that neither of the two models described here takes into account that G-proteins are also allosteric modulators of a GPCR, although a model that takes the interaction of G-proteins into account has been proposed (40). GROUPS I AND II ALLOSTERIC MODULATORS Group I In the mid-1990s, a noncompetitive antagonist for mGluR5 6-methyl2-(phenylazo)-3-pyridinol (SIB-1757), was identiﬁed in a collaborative

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high-throughput screen between SIBIA (now Merck) and Ciba-Geigy (now Novartis). SIB-1757 antagonized glutamate-induced, intracellular Ca2þ responses in an mGluR5-expressing cell line and appeared to be subtype selective for mGluR5. An additional compound from the compound libraries, (E)-2methyl-6-(2-phenylethenyl)pyridine (SIB-1893), also displayed mGluR5 antagonism (41) and based on a pharmacophore modeling approach, 2methyl-6-(phenylethynyl)-pyridine (MPEP), a potent noncompetitive antagonist, was developed. MPEP displayed nanomolar afﬁnity and a high degree of selectivity for mGluR5 over other mGluRs (42). The compound was subsequently found to bind in the 7TM domain (43), conﬁrming it as a negative allosteric modulator of mGluR5. At the same time, 7-(hydroxyimino) cyclopropan[b] chromen-1a-carboxylic acid ethyl ester (CPCCOEt) was identiﬁed as a subtype-selective noncompetitive antagonist for mGluR1 as was its close structural analog PHCCC, although PHCCC was less efﬁcacious than CPCCOEt (44). Like MPEP, CPCCOEt was found to have the proﬁle of a negative modulator, i.e., no displacement of [3H]glutamate and a binding site in the 7TM domain (45). Since the discovery of MPEP and CPCCOEt, a number of other negative and positive allosteric modulators for mGluR1 and mGluR5 have been identiﬁed. Additional allosteric modulators for Group I include the mGluR5 negative allosteric modulator 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP). This compound is a structural analog of MPEP, which unlike MPEP does not show effects on mGluR4 (46,47), the norepinephrine transporter (48), nor the NMDA receptor (49). 3,30 -Diﬂuorobenzaldazine and N-{4chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) have been identiﬁed as positive modulators of mGluR5 (50,51) and, interestingly, analogs of DFB have been shown to exert a full spectrum of allosteric modulation from negative to neutral to positive effects at the mGluR5 (50). Compounds like Ro 67–7476 are positive allosteric modulators of mGluR1 (52), while negative allosteric modulators of mGluR1 include (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methylidenhexahydro-cyclopenta[c]furan-1-on (BAY 36-7620) (53) and 1-(3,4-dihydro2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone (R214127) (Fig. 3; Table 2) (54). Group II Allosteric modulators for Group II mGluRs have also been identiﬁed. Ro 64–5229 and related compounds were found to be negative allosteric modulators of mGluR2 (55). Recently, N-(4-2-methoxyphenoxy)phenyl)-N(2,2,2-triﬂuoroethylsulfonyl)pyrid-3-ylmethylamine (LY487379) and related compounds have been identiﬁed as positive allosteric modulators for mGluR2 (56) and found to bind in the 7TM domain of mGluR2 (57)

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Figure 3 Selected mGluR allosteric modulators. Refer to the text for details. Abbreviation: mGluR, metabotropic glutamate receptor.

(Fig. 3 and Table 2). The identiﬁcation of allosteric modulators for Groups I and II and of their binding sites within the 7TM domain has provided crucial information in understanding how and where allosteric modulators of mGluRs bind. GROUP III mGluR ALLOSTERIC MODULATORS Negative Allosteric Modulators Interestingly, many of the compounds mentioned previously have a reverse allosteric effect on Group III in relation to Group I, i.e., those that are negative allosteric modulators in one group become positive in the other and vice versa (Table 2). The two mGluR5 positive allosteric modulators, DFB and CPPHA, which do not share the same binding site in mGluR5, are also weak negative allosteric modulators of mGluR4 and mGluR8 (Fig. 3 and Table 2) (50,51). Unlike DFB, CPPHA does not displace the radiolabeled MPEP analog [3H]3-methoxy-5-(2-pyridinylethynyl) pyridine, implying that the two compounds bind to different sites in the 7TM domain of the mGluR5 (51). The binding site of CPPHA and DFB in mGluR4 and 8 is not known.

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Table 2 Selected Allosteric Ligands for the mGluRs Positive allosteric modulators Group I

mGR1

mGR5 mGR2 mGR3 Group III mGR4 mGR6 mGR7 mGR8 Group II

Ro 67–7476 CPPHA, DFB LY487379 — PHCCC, SIB-1893, MPEP — — —

Negative allosteric modulators CPCCOEt, PHCCC, BAY 36–7620, R214127 MPEP, SIB-1893, MTEP Ro 64–5229 — CPPHA, DFB — — CPPHA, DFB

Note: Compounds with reverse effects at Group I versus Group III mGluRs are in bold. See text for references. Abbreviations: mGluR, metabotropic glutamate receptor; MPEP, 2-methyl-6-(phenylethynyl)pyridine; MTEP, 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine; DFB, 3,30 -diﬂuorobenzaldazine; PHCCC, N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxamide; CPPHA, N-{4chloro-2-[(1,3-dioxo-1, 3-dihydro-2H-isoindol-2-yl) methyl] phenyl}-2-hydroxybenzamide; BAY 36-7620, (3aS,6aS)-6a-naphtalan-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan1-on; SIB-1893, (E)-2-methyl-6-(2-phenylethynyl)-pyridine; CPCCOEt, 7-(hydroxyimino) cyclopropa[b]chromen-1a-carboxylate ethyl ester. R214127, 1-(3,4,-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone; LY487379, N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2triﬂuoroethylsulfonyl)pyrid-3-ylmethylamine.

Positive Allosteric Modulators of mGluR4 MPEP and SIB-1893 MPEP and SIB-1893, well-characterized mGluR5 allosteric antagonists (41,42), have been found to increase both the efﬁcacy and the potency of glutamate as well as L-AP4 on the human mGluR4 (58). In a [35S]GTPcS binding assay performed on membranes prepared from mGluR4 expressing baby hamster kidney cells, MPEP increased both the efﬁcacy and potency of L-AP4 and was devoid of effects when applied alone at concentrations up to 100 mM (Fig. 4). The maximal efﬁcacy of L-AP4 increased from 1.9- to 2.9-fold above basal [35S]GTPcS binding whereas its potency increased twofold in the presence of 100 mM MPEP. Moreover, the Group III orthosteric antagonist (RS)-a-cyclopropyl-4-phosphonophenylglycine (CPPG) completely abolished the effect of MPEP, showing that positive modulation of MPEP was fully dependent on orthosteric receptor activation by L-AP4 in [35S]GTPcS binding (Fig. 5). Taken together, these data suggest that MPEP increases the sensitivity of mGluR4 toward orthosteric agonists. Similar [35S]GTPcS binding data were obtained for SIB-1893, although SIB-1893 increased the efﬁcacy and potency of the orthosteric agonists even further [see Mathiesen et al. (58)].

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Figure 4 Effect of MPEP- on L-AP4–induced [35S]GTPcS binding to mGluR4 expressing BHK cell membranes. Data are given as the mean SEM from a representative experiment where each data point was determined in triplicate. At least three independent experiments showing the same results were performed. For similar data on SIB-1893 see Mathiesen et al. (58). Abbreviations: MPEP, 2-methyl-6-(phenylethynyl)-pyridine; L-AP4, L-(þ)-2-amino-4-phosphonobutyric acid; mGluR, metabotropic glutamate receptor; BHK, baby hamster kidney.

Since MPEP and SIB-1893 did not increase [35S]GTPcS binding above basal levels when applied alone, and since their potentiating effects rely on orthosteric activation of the receptor, it indicated an allosteric site of action for these compounds. To conﬁrm this, radioligand binding studies on mGluR4-expressing membranes were performed. Unlike cold L-AP4, neither MPEP nor SIB-1893 displaced the orthosteric radioligand [3H]LAP4 (20 nM) from its binding site in the ATD, conﬁrming their allosteric site of action (Fig. 6). In addition, SIB-1893 increased the speciﬁc binding of [3H]L-AP4 for mGluR4, which is in agreement with the allosteric modulation models of Hall (38) and Parmentier (39). Since MPEP did not have this effect, it appears that SIB-1893 is a more potent mGluR4 allosteric modulator than MPEP. This observation is substantiated in further [35S]GTPcS and cAMP studies. mGluR4 has been shown to inhibit forskolin-stimulated cAMP production through activation of Gai. In order to examine the effect of MPEP and SIB-1893 in a whole cell system, L-AP4 concentration–response curves in the presence of MPEP and SIB-1893 were generated. As shown in Figure 7, L-AP4 alone produced a concentration-dependent inhibition of forskolinstimulated cAMP with an EC50 of 160 nM (pEC50 ¼ 6.80 0.17) and maximal efﬁcacy of 56 2%. In the presence of MPEP and SIB-1893, the apparent potency and the maximal efﬁcacy of L-AP4 was augmented (latter indicated by the arrows) as seen in the [35S]GTPcS binding studies (Fig. 5). At 10 mM,

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Figure 5 Effect of the orthosteric antagonist CPPG on MPEP potentiation of 35 L-AP4–induced [ S]GTPcS binding to mGluR4 expressing BHK cell membranes. CPPG antagonized the submaximal L-AP4 response (0.8 mM). The dose-dependent increase in L-AP4 activity by MPEP was completely abolished in the presence of CPPG. The data are given as the mean SEM from three to six independent experiments each performed in quadruplicate. Abbreviations: MPEP, 2-methyl-6(phenylethynyl)-pyridine; L-AP4, L-(þ)-2-amino-4-phosphonobutyric acid; mGluR, metabotropic glutamate receptor; BHK, baby hamster kidney; CPPG, (RS)-a-cyclopropyl-4-phosphonophenylglycine.

MPEP and SIB-1893 lowered the EC50 values of L-AP4 threefold to 54 nM (pEC50 ¼ 7.27 0.20) and fourfold to 38 nM (pEC50 ¼ 7.42 0.29), respectively. Additionally, the ability of L-AP4 to inhibit forskolin-stimulated cAMP production was increased to 46 2% for both compounds. However, unlike the [35S]GTPcS binding studies, MPEP and SIB-1893 appeared to have endogenous effects on the inhibition of forskolin-stimulated cAMP production (Fig. 7). It is important to note that MPEP and SIB-1893 were devoid of endogenous effects on forskolin-stimulated cAMP production in parental and in mGluR2 expressing cell lines (data not shown). To further characterize these endogenous effects, concentration– response curves of MPEP and SIB-1893 were generated and compared to L-AP4 (Fig. 8A). Without addition of orthosteric agonist, MPEP and SIB-1893 activated the receptor with EC50 values of 87 mM (pEC50 ¼ 4.06 0.23) and 32 mM (pEC50 ¼ 4.49 0.16), respectively, as compared to the L-AP4 EC50 value of 0.16 mM (pEC50 ¼ 6.80 0.17). The maximal efﬁcacies of MPEP (42 8%) and SIB-1893 (30 5%) to inhibit forskolinstimulated cAMP production were above that of L-AP4 (56 2%). Since this endogenous effect was lowered in the presence of the orthosteric antagonist CPPG and since CPPG itself caused an increase in cAMP above

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Figure 6 Lack of displacement of [3H]L-AP4 by MPEP and SIB-1893 in mGluR4 expressing membranes. The orthosteric agonist [3H]L-AP4 (20 nM) is displaced from mGluR4 expressing membranes by its cold homolog L-AP4 and not by SIB-1893 and MPEP. This conﬁrmed the allosteric site of action by MPEP and SIB-1893. Note that increasing concentrations of SIB-1893 but not MPEP appear to increase the speciﬁc binding of [3H]L-AP4 to mGluR4. Data are given as the mean percent of speciﬁc [3H]L-AP4 binding SEM from a representative experiment performed in triplicate. Four independent experiments with similar results were performed. Signiﬁcant differences from speciﬁc [3H]L-AP4 binding: P < 0.01 (ANOVA followed by Dunnett’s test). Binding experiments were performed using SPA-beads as described previously. Abbreviations: mGluR, metabotropic glutamate receptor; L-AP4, l-(þ)-2amino-4-phosphonobutyric acid; ANOVA, analysis of variance; MPEP, 2-methyl6-(phenylethynyl)-pyridine; SIB-1893, (E)-2-methyl-6-(2-phenylethynyl)-pyridine. Source: From Ref. 58.

that of forskolin alone, it was concluded that the endogenous effect of the compounds were due to mGluR4 preactivation (Figs. 8B and C). Receptor preactivation may be due to either the presence of glutamate in the assay media or the possibility that the mGluR4 in this expression system is constitutively active. Since a lowering of the basal activity by CPPG was not observed in the membrane-based [35S]GTPcS assay (Fig. 5), these data point toward receptor activation due to glutamate. It was recently reported, however, that a weak endogenous effect of the mGluR4 positive allosteric modulator PHCCC in a [35S]GTPcS assay performed on mGluR4 membranes might be due to constitutive activity (32). Interestingly, mGluR1 and mGluR5 have been shown to exhibit endogenous constitutive activity in neurons, which appears to be regulated by direct interaction with the homer proteins (59). Thus, the possibility that mGluR4 in our expression system is constitutively activated by a similar mechanism, which is only functional in a whole-cell assay, cannot be excluded. Gai-coupled receptors can activate PLC via chimeric G-proteins when the last ﬁve amino acids of Gaq are exchanged with those of Gao (60).

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Figure 7 Effect of (A) MPEP and (B) SIB-1893 on L-AP4-induced inhibition of forskolin stimulated cAMP-formation in BHK cells expressing mGluR4. Data are given as the mean percent of forskolin stimulated cAMP production SEM from three to seven independent experiments each performed in triplicate. Abbreviations: MPEP, 2methyl-6-(phenylethynyl)-pyridine; cAMP, cyclic adenosine monophosphate; BHK, baby hamster kidney; mGluR, metabotropic glutamate receptor; SIB-1893, (E)-2methyl-6-(2-phenylethynyl)-pyridine.

In order to evaluate the effects of SIB-1893 and MPEP in another whole-cell system, HEK cells stably expressing the mGluR4 were transiently transfected with the chimeric Gaqo5 and assayed for intracellular Ca2þ mobilization. The effects of SIB-1893 and MPEP were compared with the recently reported mGluR4 positive allosteric modulator PHCCC (32) in this assay (27). In these studies, 100 mM L-AP4 robustly mobilized intracellular Ca2þ, and the simultaneous application of L-AP4 (100 mM) and any of the positive allosteric modulators SIB-1893 (100 mM), MPEP (100 mM), or

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Figure 8 Effect of MPEP and SIB-1893 alone and in presence of 100 mM CPPG in BHK cells expressing mGluR4. (A) Concentration–response curves for inhibition of forskolin stimulated cAMP-formation by the orthosteric agonists L-AP4 and modulators. Effect of 100 mM CPPG on (B) MPEP and (C) SIB-1893 concentration– response curves. Data are expressed as the mean percent of forskolin-stimulated cAMP production SEM from three independent experiments, each performed in triplicate. Abbreviations: MPEP, 2-methyl-6-(phenylethynyl)-pyridine; BHK, baby hamster kidney; mGluR, metabotropic glutamate receptor; cAMP, cyclic adenosine monophosphate; L-AP4, l-(þ)-2-amino-4-phosphonobutyric acid; SIB-1893, (E)-2methyl-6-(2-phenylethynyl)-pyridine; CPPG, (RS)-a-cyclopropyl-4-phosphonophenylglycine.

PHCCC (10 mM) signiﬁcantly increased the maximal response of L-AP4. CPCCOEt (10 mM), which is not a positive allosteric modulator of mGluR4 despite its close structural resemblance to PHCCC, did not show any effects when coapplied with L-AP4 (Fig. 9). Minor effects of SIB-1893, MPEP, and PHCCC, but not of CPCCOEt, were observed when applied alone. However, the endogenous efﬁcacies for SIB-1893 and MPEP were lower than in the cAMP assay (Fig. 8A). Nevertheless, these experiments demonstrated that positive allosteric modulation of mGluR4 could be reproduced in

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Figure 9 Effect of mGluR4 positive allosteric modulators on the maximal efﬁcacy of L-AP4 in a intracellular Ca2þ mobilization assay. A stable mGluR4 expressing HEK cell line was transiently transfected with the chimeric G-protein Gaqo5 in order to make the normally Gai coupling mGluR4 capable of activating PLC. The mGluR4 positive allosteric modulators SIB-1893, MPEP, and PHCCC had minor effects when applied alone but signiﬁcantly increased the maximal efﬁcacy of the orthosteric agonist L-AP4. CPCCOEt, previously shown to have no effect on mGluR4 despite its close structural resemblance to PHCCC, was used as a negative control. The data are given as the mean SEM from three independent experiments, each performed in sextuplicate. Signiﬁcant differences from L-AP4 maximal response, when allosteric modulators were incubated in the presence of L-AP4: P < 0.01 (ANOVA followed by Dunnett’s test). Abbreviations: mGluR, metabotropic glutamate receptor; L-AP4, L-(þ)-2-amino-4-phosphonobutyric acid; ANOVA, analysis of variance; MPEP, 2-methyl-6-(phenylethynyl)-pyridine; SIB-1893, (E)-2methyl-6-(2-phenylethynyl)-pyridine; PHCCC, N-phenyl-7-(hydroxyimino) cyclopropa [b]chromen-1a-carboxamide; CPCCOEt, 7-(hydroxyimino)cyclopropa[b]chromen-1acarboxylate ethyl ester.

another whole-cell functional assay and that the endogenous modulator efﬁcacy was dependent on the assay used. Possible explanations for the lower endogenous effects of SIB-1893 and MPEP could be due to the transient nature of Ca2þ mobilization or to a higher degree of receptor preactivation in the cAMP assay. In the Ca2þ mobilization assay, the maximal response is measured as the maximal change in intracellular Ca2þ, which is normally achieved eight seconds after agonist application, while the cAMP accumulation is measured over 15 minutes. During the longer 15-minute assay time, ambient glutamate concentrations may rise due to further glutamate release from the cells.

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The presence of glutamate in whole-cell assays and its effects on mGluR pharmacology have been previously reported (61), which poses the challenge of classifying SIB-1893 and MPEP as either allosteric modulators or allosteric agonists in systems where glutamate is ever present. In addition, pharmacological studies of MPEP and SIB-1893 as mGluR4 positive allosteric modulators are likely to be obscured as a consequence of their antagonistic effects on mGluR5 (62). Care should be taken to use models where mGluR5 is not expressed such as in mGluR5 or mGluR4 knockout mice when making conclusions about the actions of MPEP and SIB-1893. Perhaps, a combined effect of mGluR5 antagonism and mGluR4 activation would prove beneﬁcial in a number of diseases and would be preferred. PHCCC PHCCC, which was ﬁrst identiﬁed as an mGluR1 antagonist (44), was recently reported to be a positive allosteric modulator of mGluR4 (27,32). The compound was found to increase both the potency and maximal efﬁcacy of glutamate (32) and L-AP4 (27) in [35S]GTPcS binding and functional assays with all activity residing in the ()-PHCCC enantiomer (32). Interestingly, a modest endogenous effect of PHCCC was found in a [35S]GTPcS binding assay performed on stable mGluR4 expressing membranes. At other mGluR subtypes, PHCCC had either no effect or weak antagonism, making it the most selective mGluR4 positive allosteric modulator to date. Unlike SIB-1893 and MPEP, PHCCC has been shown to positively modulate mGluR4 in native systems and have effects in models of Parkinson’s disease and neuroprotection. In electrophysiological studies on the striatopalliday synapse, presynaptical mGluR4 was shown to produce inhibition of GABAergic transmission (27). Furthermore, PHCCC had no effect alone, but instead potentiated L-AP4-induced inhibition of synaptic transmission in agreement with a role as a positive allosteric modulator. At synapses expressing mGluR7 and -8, PHCCC did not potentiate L-AP4–induced inhibition of synaptic transmission (27). In models of NMDAand b-amyloid–induced neurotoxicity, ()-PHCCC was shown to potentiate neuroprotection induced by L-AP4, although it also had a neuroprotective effect alone. This was ascribed to the fact that extracellular glutamate concentration in the medium increased during NMDA challenge. In support of this, the neuroprotective effect of ()-PHCCC alone was reversed by the application of orthosteric Group III antagonists (32). Finally, PHCCC showed an antiparkinsonian effect in a model of Parkinson’s disease in rats when applied alone, suggesting that selective activation of mGluR4 by an allosteric modulator has therapeutic potential (27). Summary of mGluR4 Modulators and Their Effects at Group I Interestingly, the rank order of SIB-1893 and MPEP on mGluR4 activity is the reverse of their negative effects on mGluR5, where MPEP is a more

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potent allosteric antagonist than SIB-1893. It is noteworthy that the other identiﬁed mGluR4 positive allosteric modulator PHCCC is an allosteric antagonist at mGluR1. CPCCOEt, a very close structural analog of PHCCC and also an allosteric antagonist of mGluR1, is completely devoid of effects at mGluR4 (27). Thus, the compounds identiﬁed as positive allosteric modulators at mGluR4 are negative allosteric modulators at Group I mGluRs. While the binding site of mGluR4 positive allosteric modulators remains to be identiﬁed, MPEP and CPCCOEt have been found to bind to the same regions in the 7TM domain, but interact with different nonconserved amino acid residues (43). Presumably, mGluR4 positive allosteric modulators bind in the same regions in the 7TM domain. It is conceivable that MPEP and SIB-1893 at mGluR5 and PHCCC at mGluR1 stabilize an inactive conformation, whereas they stabilize an active conformation at mGluR4. The observation that the mGluR5 positive allosteric modulator DFB partially displaces an MPEP analog from its binding site on mGluR5 and is a negative allosteric modulator of mGluR4 further supports the idea of a common binding region for allosteric modulators in the 7TM domain of mGluRs. However, additional binding domains in the 7TM region may exist since the mGluR5 positive allosteric modulator CPPHA does not displace an MPEP analog from mGluR5 but still negatively modulates mGluR4.

POTENTIAL MECHANISTIC EFFECTS OF A GROUP III POSITIVE ALLOSTERIC MODULATOR An interesting question is how a Group III positive allosteric modulator mechanistically behaves in vivo. Studies of the mGluR4 positive allosteric modulator PHCCC and the mGluR2 positive allosteric modulator LY487379 provide some clues. Besides presynaptical terminals at glutamatergic synapses mGluR4 is localized to GABAergic synapses, where it is believed to function as a heteroautoreceptor of GABAergic neurotransmission. Here, PHCCC was without effect when applied alone (27), which is most likely due to a lack of diffusion of glutamate to the GABAergic synapse. Likewise, the mGluR2 positive allosteric modulator LY487379 was tested for allosteric modulation of mGluR2 in the medial perforant path–dentate gyrus synapse where Group II activation has been shown to inhibit glutamatergic transmission (57). Like PHCCC, LY487379 showed minimal effects alone but signiﬁcantly potentiated the inhibitory effect of a Group II speciﬁc agonist. These ﬁndings suggest that the mGluR2 in this hippocampal slice preparation was not activated by synaptically released glutamate when stimulated at low frequencies (57). Thus, the two described electrophysiological studies with the positive allosteric modulators of mGluR4 and mGluR2 suggest that glutamate in the brain-slice preparations tested are not present in sufﬁcient amounts to

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activate the respective receptors, which could be due to a lack of excitatory drive from other synapses absent in the respective preparations (27). However, there are several reports on tonic activation and thus tonic autoinhibition of excitatory postsynaptic potentials (EPSPs) by Group III mGluRs in glutamatergic synapses. In the hippocampus, there is evidence of persistently active mGluRs that cause a reduction in the ﬁrst EPSPs after application of a high-frequency stimulus, which is not enhanced by additional stimuli (63). Furthermore, an increase in EPSPs in rat visual cortex has been observed after application of Group III antagonists (64). Recently, it was found that CPPG, besides inhibiting L-AP4-induced depression of spontaneous glutamate- and GABA-mediated synaptic currents, increased synaptic activity recorded in hypocretin neurons in hypothalamic slices on its own (65). In vivo microdialysis studies in rat nucleus accumbens support the existence of Group III mGluR basal tone by extracellular glutamate together with regulation of nonvesicular glutamate release by Group III mGluRs (66). On the other hand, CPPG in the cerebellar cortex only had endogenous effects after prolonged stimulus trains, which most likely leads to the release of endogenous glutamate (67). This was also the case for another Group III antagonist M-AP4 after an EPSP was evoked by electrical stimulation (68). Together, these data suggest that at least in some regions of the CNS, Group III mGluRs are tonically activated under normal/basal conditions by ambient glutamate while other mGluRs may only function as autoinhibitory receptors after initial glutamate release from the presynaptic neuron. Given this apparent preactivation of mGluR4 in some (presumably glutamatergic) synapses as described above, one may speculate on the nature of a positive allosteric modulator in an in vivo environment. Regardless of the origin of mGluR4 preactivation in the study of MPEP and SIB-1893 as positive allosteric modulators of mGluR4 (58), the endogenous effects of SIB-1893 and MPEP in the cAMP assay of this study (Figs. 7 and 8A) could well reﬂect their in vivo effects. Indeed, the mGluR4 positive allosteric modulator PHCCC showed in vitro and in vivo effects when applied alone (32). Another aspect that should be considered is the fact that the pharmacological properties of the positive allosteric modulator may inﬂuence the ﬁnal effect of a positive allosteric modulator. A positive allosteric modulator may increase potency or efﬁcacy, or both, of the orthosteric agonist for the receptor. Examples of allosteric modulators that increase both potency and efﬁcacy of an orthosteric agonist are MPEP, SIB-1893, and PHCCC for mGluR4 (26,32,58). Interestingly, the recently described mGluR5 positive allosteric modulators DFB and CPPHA only increased potency but not efﬁcacy of an orthosteric agonist in a recombinant expression system (50,51). If not for these compounds, one might conclude that the increase of efﬁcacy is merely a receptor overexpression phenomenon whereas the increase in potency is a ‘‘true’’ positive allosteric modulator effect. For the mGluR2 positive allosteric modulator LY487379, it has been suggested that the

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increase in efﬁcacy of the orthosteric agonist is an overexpression phenomenon due to spare receptors (57). This scenario was suggested since an increase in potency but not efﬁcacy was observed for LY487379 in native tissues, while an increase in both efﬁcacy and potency was observed when the mGluR2 was expressed in recombinant systems (57). PERSPECTIVE It is conceivable that modulation for Family C GPCRs may give rise to allosteric effects not previously described where close cooperativity between two domains is expected within a single receptor. What does this mean in terms of receptor activation and the design of therapeutics with novel modes of action? Both in vitro and in vivo studies have shown that positive allosteric modulators of mGluR4, in most cases, have an endogenous effect, probably due to the presence of ambient glutamate or a glutamatergic tone keeping the receptor in a more or less preactivated state. One of the proposed beneﬁcial effects of positive allosteric modulation is that an allosteric modulator will only exert its effect on the receptor by enhancing the orthosteric agonist response when the orthosteric agonist is present in the synapse. This means that when the modulator is present in the synapse alone, there is no receptor activation and thus receptor desensitization and downregulation will be reduced compared to treatment with an orthosteric agonist. However, it can be questioned whether this will also be the case for a presynaptically located Group III receptor where there is glutamatergic tone. In synapses with glutamatergic tone, the presence of a positive allosteric modulator could lead to persistent receptor activation as observed for orthosteric agonists. However, the likelihood of receptor downregulation and desensitization may be smaller for a positive allosteric modulator as well as in synapses with no glutamatergic tone. As mentioned before, there is evidence for a role of Group III mGluRs in epilepsy as well as in maintaining glutamate homeostasis and in mediating excitotoxicity. Since upregulation of mGluR4 most likely serves as an autoregulatory mechanism to counteract the enhanced excitatory activity observed in epilepsy and neurodegeneration, a positive allosteric modulator of mGluR4, which increases the maximal efﬁcacy of an orthosteric agonist, seems very attractive in these pathophysiological conditions. In the quest for developing therapeutics that target diseases where glutamate dysfunction is at the root of the problem, a detailed understanding of the mechanism of allosteric modulation is crucial. Analogous to the ‘‘power on’’ knob on a radio, glutamate serves to turn on mGluRs. But what may be even more important is the ability to control the volume of the music to varying degrees. In essence, that is precisely what allosteric modulators have the potential to do. Obviously, there is a need to develop a volume control system for the ﬁne-tuning of this radio.

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10 Allosteric Modulation of GABAB Receptors Stephan Urwyler Department of Neuroscience, Novartis Institutes for BioMedical Research, Basel, Switzerland

INTRODUCTION: STRUCTURE AND FUNCTION OF THE GABAB RECEPTOR c-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS). A great number of major CNS-active, investigational, recreational, or clinically used drugs exert their effects through the GABAergic system. The antiepileptic drugs tiagabine (a GABA-transport blocker) and vigabatrine (c-vinyl GABA, a GABAtransaminase inhibitor) both act by increasing GABA concentrations in the nervous tissue. However, most pharmacologically active compounds interacting with GABAergic neurotransmission are targeted at the receptors for GABA (1,2). The hallucinogenic and CNS-depressant drug muscimol is a selective agonist at the GABAA receptor, a ligand-gated ion channel, which by enabling the inﬂux of chloride ions hyperpolarizes the postsynaptic neuronal membrane. On the other hand, the convulsant drugs bicuculline and picrotoxin inhibit this receptor. Barbiturates (such as pentobarbital) and benzodiazepines (diazepam, chlordiazepoxide) are in clinical use as anesthetic, anxiolytic, anticonvulsant, and muscle-relaxant agents. Both classes of drugs enhance the chloride conductance of the GABAA receptor, albeit through different mechanisms. Most barbiturates prolong the open time of the chloride channel also in the absence of GABA, whereas the 235

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benzodiazepines increase its opening frequency only when GABA is bound to the receptor (see also Chapter 4). Alcohol as well as a number of anesthetic agents, such as propofol or halothane, are also known to enhance the function of GABA receptor–gated chloride channels. Lioresal1, a racemic mixture of b-p-chlorophenyl-GABA (baclofen), a lipophilic, brain-penetrating analog of GABA, was ﬁrst marketed in the beginning of the 1970s as a muscle-relaxant drug used in the treatment of spasticity arising from multiple sclerosis or spinal injury. It was only much later that baclofen was found to interact stereospeciﬁcally with a novel type of GABA receptor. In fact, its inhibitory effects on the release of different neurotransmitters in distinct tissue preparations were found to be bicucullineinsensitive, whereas the bicuculline-sensitive GABA receptor agonist 3aminopropane sulphonic acid did not affect neurotransmitter release (3). The existence of this novel, distinct ‘‘GABAB’’ receptor was soon thereafter conﬁrmed in radioligand-binding experiments (4). In the subsequent decades, a great number of useful pharmacological tools, potent and selective agonists and antagonists, for the GABAB receptor became available [for review, see Bowery et al. (2)]. Also, a multitude of biochemical and physiological studies made it clear that this receptor, like the GABAA receptor, was of inhibitory nature, but unlike the former, it coupled to G-protein– mediated intracellular pathways. However, while the existence and the properties of this GABAB receptor were well established on pharmacological and physiological grounds, its molecular entity remained elusive for a long time. It was not until 1997 that a complementary DNA, encoding a quite large (ca 100 kDa) GABAB receptor protein, was successfully cloned for the ﬁrst time (5). It was found to resemble metabotropic glutamate receptors (mGluRs) and exist in two distinct variants, named GABAB(1a) (containing 960 amino acids) and GABAB(1b) (844 amino acids), which differ in the presence or the absence, respectively, of an N-terminal complement protein sequence (Sushi repeats). These isoforms are not splice variants, but arise from the initiation of transcription at different sites, under the control of distinct promoters. However, true splice variants of the GABAB(1) protein have also been shown to exist [summarized in Refs. (6–8)]. Surprisingly, although heterologous expression of either GABAB receptor protein allowed measuring the binding of high-afﬁnity antagonist radioligands, a considerably lower binding afﬁnity for agonists compared to native receptors was found, and it did not make possible the measurement of robust functional responses. This ﬁnding remained enigmatic until several groups reported at the same time the cloning of a second GABAB receptor protein (GABAB2) containing 941 amino acids (110 kDa) (9–12). GABAB(1) and GABAB(2) are derived from different genes and share 35% sequence identity and about 50% similarity. The coexpression of GABAB(1) and GABAB(2) to form heterodimeric assemblies, a unique feature among Gprotein–coupled receptors (GPCRs), is a prerequisite to form functional

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GABAB receptor entities. This breakthrough discovery has fostered intense research efforts in recent years, which have greatly increased our knowledge of the structure and function of the GABAB receptor (8,13). Both GABAB receptor proteins are part of the ‘‘Family 3’’ or ‘‘Class C’’ GPCRs, which also comprises the phylogenetically related mGluRs, the calcium-sensing receptor, and mammalian odorant and taste receptors (14–16). Like the other receptors that belong to this family, GABAB proteins have a molecular structure (Fig. 1), which is characterized by its seven transmembrane-spanning domains, an intracellular C-terminus apt to interact with other proteins, and an unusually large extracellular N-terminal ligandbinding domain related to bacterial periplasmic amino acid–binding proteins (17,18). Unlike other Family 3 GPCRs, the GABAB receptor does not have an extracellular cysteine-rich sequence between the ligand-binding and the transmembrane domains. The ligand-binding domain is made up of two large globular lobes; agonist binding induces a hinge-bending conformational change,

Figure 1 Schematic representation of the structure of the GABAB receptor. The receptor consists of a heterodimeric assembly of two distinct subunits, GABAB(1) and GABAB(2). The ligand-binding domain is formed by two lobes in GABAB(1) which close on agonist binding in a manner reminiscent of the Venus ﬂy trap plant, thereby triggering signal transduction. Competitive antagonists bind to the same site, without closing the lobes of the VFTM. The homologous part in GABAB(2) is unable to bind orthosteric ligands. On the other hand, it is the GABAB(2) subunit that couples to G-proteins with their a, b, and c-subunits, thereby inhibiting the formation of cAMP, the opening of presynaptic voltage-sensitive calcium channels, or activating postsynaptic inwardly rectifying potassium channels (Kir 3). Moreover, the GABAB(2) subunit is essential for the expression of GABAB(1) at the cell surface by masking a retention signal in the C-terminal part of GABAB(1) via a coiled–coil interaction of the intracellular parts of the two subunits. Abbreviations: GABAB, c-Aminobutyric acid B; cAMP, cyclic adenosine monophosphate; VFTM, Venus ﬂy trap module.

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closing these lobes and trapping the agonist between them, in a manner reminiscent of the mode of insect trapping of the carnivorous ‘‘Venus ﬂy trap’’ plant (19–23). Although the GABAB(2) subunit also contains this Venus ﬂy trap module (VFTM) (Fig. 1), it cannot bind GABA or other agonist or antagonist ligands, because amino acids in the ﬂy trap module of GABAB(1), which are critical for ligand binding, are not conserved in GABAB(2) (21). Despite the inability of the GABAB(2) subunit to bind orthosteric ligands (agonists, competitive antagonists), it is essential for GABAB receptor expression and function in several ways. GABAB(1), when expressed alone, is not incorporated into the plasma membrane, because a retention sequence at its C-terminal end prevents its release from the endoplasmatic reticulum. The interaction of the C-terminal parts of GABAB(1) and GABAB(2), forming a ‘‘coiled coil domain,’’ masks this retention signal, thus making possible the transport of the heterodimeric complex to the cell surface. GABAB(2), on the other hand, does not need this interaction with GABAB(1) for its incorporation into the plasma membrane. Moreover, it has been shown that the extracellular N-terminal domain of GABAB(2) interacts allosterically with its GABAB(1) counterpart, thus facilitating agonist binding and conferring to it the afﬁnity that the native GABAB receptor has for agonists (24). Finally, whereas receptor activation is initiated by agonist binding to the VFTM of GABAB(1), it is the GABAB(2) subunit that transmits the signal to the intracellular effector systems via an interaction of its second intracellular loop with the appropriate G-proteins (25–28). Native and recombinant GABAB receptors couple to various intracellular effector systems. Baclofen and other GABAB receptor agonists produce a robust inhibition of forskolin-stimulated adenylyl cyclase activity in rat brain tissue preparations (29–31) and in mammalian cell lines heterologously expressing the GABAB receptor (32,33). The GABAB receptor– mediated inhibition of cyclic adenosine monophosphate (cAMP) formation is pertussis toxin sensitive (34), indicating the involvement of Gi - and/or Go-protein a-subunits. On the other hand, baclofen has been reported to enhance the activity of adenylyl cyclase stimulated by corticotropinreleasing hormone (CRH), pituitary adenylate cyclase-activating peptide, or noradrenaline in native systems (31,35,36). These effects are most likely due to receptor–receptor interactions (‘‘crosstalk’’) and seem, like the stimulation of basal adenylyl cyclase activity, to involve signaling by b,c–G-protein subunits (35,36). Also, via this pathway, GABAB receptors are linked to G-protein coupled inwardly rectifying potassium channels (Kir3-type, also known as GIRK channels) (37). The activation of these channels via GABAB receptor stimulation results in inhibition of neuronal excitability by producing a late, long-lasting component of the inhibitory postsynaptic potentials (IPSPs). The ionotropic GABAA receptor, on the other hand, mediates the fast, short-lasting component of IPSPs. Furthermore, presynaptic GABAB auto- and heteroreceptors inhibit the depolarization-induced release of a

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number of neurotransmitters, such as GABA itself or various neuropeptides, catecholamines, acetylcholine, or the excitatory amino acid glutamate (3,38– 42). The mechanism responsible for these presynaptic effects of GABAB receptors seems to involve the inhibition of voltage-sensitive calcium channels (43). In light of the importance of the GABAergic system in CNS physiology and pathophysiology, and the multitude of functions of the GABAB receptor in synaptic transmission, it may seem surprising that to date, baclofen is the only therapeutically used drug acting through GABAB receptors. There seems to remain a great potential for this receptor as a drug target. In light of the clinical success of benzodiazepines as allosteric enhancers of the ionotropic GABAA receptor, it appears that, in particular, agents acting in the same way at the metabotropic GABAB receptor should also be appealing novel pharmaceuticals. Allosteric modulators act synergistically with an agonist, usually without activating a receptor on their own. Thus, they act in concert with physiological neurotransmission in its temporal and spatial organization, in contrast to agonists, such as baclofen, that activate receptors independently of synaptic activity. Therefore, positive modulators are expected to have a better side effect proﬁle and a lower propensity for receptor downregulation (tolerance development) on chronic treatment than agonists. For these reasons, the concept of allosteric modulation of GPCRs as a therapeutic principle has recently attracted considerable interest (44–48). It is against this background that we have set out to ﬁnd novel compounds that act with novel mechanisms at the GABAB receptor. THE DISCOVERY OF ALLOSTERIC GABAB RECEPTOR MODULATORS Modern assay technologies, in conjunction with the stable heterologous expression of a drug target of interest in recombinant cell lines, make it possible to screen large compound libraries containing hundreds of thousands of chemical entities for new pharmacological actions. One assay that is frequently used to assess GPCR function measures the stimulation of guanosine 50 -O-3-thiotriphosphate [GTP(c)35S] binding in cell membrane preparations. The ﬁrst step in the signal transduction pathways stimulated by GPCRs is the exchange of guanosine diphosphate by guanosine-50 -Otriphosphate (GTP) at the a-subunit of the respective G-proteins. If GTP is replaced by its metabolically stable analog GTP(c)S, this step can easily be measured by assessing the accumulation of GTP(c)35S in its radioactive form. It is with such a GTP(c)35S assay using membranes from a Chinese hamster ovary (CHO) cell line stably expressing the GABAB receptor that the two positive allosteric modulators 2,6-Di-tert-butyl-4-(3-hydroxy-2,2dimethyl-propyl)-phenol (CGP7930) and N,N0 -Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) (Fig. 2) have been discovered (49,50). These two compounds enhance the stimulation of GTP(c)35S

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Figure 2 Chemical structures of CGP7930 (left) and GS39783 (right).

binding by GABA or baclofen without activating it by themselves (Fig. 3). Interestingly, CGP7930 is a close derivative of the general anesthetic propofol, which enhances agonist-induced activation of the GABAA receptor, but was found to be completely devoid of any such effect on the GABAB receptor. On the other hand, the aldehyde analog of CGP7930 (CGP13501) and a number of close derivatives of GS39783 were also found to enhance GABAB receptor–mediated GTP(c)35S binding, although being weaker than the two prototype compounds (49,50). We observed no enhancement of GABA-stimulated GTP(c)35S binding by CGP7930 or GS39783 in the presence of the competitive GABAB receptor antagonist CGP56999A, further conﬁrming that the effects of these compounds are dependent on the simultaneous activation of the receptor by an agonist.

Figure 3 Concentration–response curves for GABA in the GTP(c)35S binding assay in the absence [&] and in the presence of CGP7930 (, 1 mM; ^, 3 mM; D, 10 mM; , 30 mM). GABA responses were measured using a recombinant GABAB receptor preparation from a stably transfected CHO cell line. The EC50 value for GABA was decreased from 4.9 mM in the absence of the modulator to 0.8 mM in the presence of 30 mM CGP7930. Abbreviations: GABA, c-Aminobutyric acid; GTP(c)35S, guanosine 50 -O-3-thiotriphosphate; CHO, Chinese hamster ovary. Source: From Ref. 49.

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One major advantage of the GTP(c)35S binding assay, from a mechanistic point of view, is that it measures the very ﬁrst step in GPCR signal transduction, and thus reﬂects an event that is very close to receptor activation. Moreover, the use of recombinant cell lines easily allows performing adequate control experiments by testing compound effects on cells that have not been transfected with the receptor of interest. CGP7930 and GS39783 did not enhance the stimulation of GTP(c)35S binding induced by glutamate in membranes from a CHO cell line expressing the mGluR2. Since this is a receptor from the same GPCR family, coupling to the same G-proteins as GABAB receptors and expressed in the same host cell line, this control experiment allows the conclusion that the enhancing effects of CGP7930 and GS39783 do not take place at the level of the cell membrane or the G-protein, but must occur at the receptor itself, indicating that they are of allosteric nature. EFFECTS OF ALLOSTERIC MODULATORS AT NATIVE GABAB RECEPTORS Despite the great value of recombinant cell lines, it was of course important to demonstrate that CGP7930 and GS39783 have the same enhancing effects on the function of native GABAB receptors. We achieved this by demonstrating the enhancement of GABAB receptor–mediated GTP(c)35S binding by CGP7930 and GS39783 in rat brain cortical membranes (49,50). The effects of both modulators at native and recombinant GABAB receptors were very similar. Also, increases in agonist-binding afﬁnities in the presence of the two modulators were found in radioligand-binding experiments with native GABAB receptors in rat brain tissue (49–51). Onali et al. (52) have subsequently conﬁrmed the allosteric effects of CGP7930 on native GABAB receptors by cAMP measurements in membranes from different rat brain regions. Their experiments comprised the study of basal and forskolin-, CRH-, and Ca2þ/calmodulin–stimulated adenylyl cyclase activity and thus demonstrated that both signaling pathways via the a- and the b/c- subunits of Gi/o-proteins are affected by the allosteric modulator. More recently, Olianas et al. (53) have reported allosteric effects of CGP7930 on GABAB receptors in samples from human postmortem brain tissue, using GTP(c)35S and cAMP assays. MOLECULAR MECHANISMS AND SITE OF ACTION OF ALLOSTERIC GABAB RECEPTOR MODULATION BY CGP7930 AND GS39783 A striking characteristic of the potentiating effects of CGP7930 and GS39783 on agonist stimulation of native and recombinant GABAB receptors is the ﬁnding that both the potency and the maximal efﬁcacy of GABA are enhanced (Fig. 3). While the effective concentration 50 (EC50)-values for

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GABA were decreased 5- to 10-fold in the presence of maximally active concentrations of the two modulators, the maximal stimulation of GTP(c)35S binding at saturating GABA concentrations was 1.5–twofold of that obtained under control conditions (in the absence of modulators) (49,50). Such a dual action was also observed by others in adenylyl cyclase assays (52,53) as well as by us by using various other experimental systems (see later). It is unlike that of most other allosteric receptor modulators known from the literature (such as the benzodiazepines), which only enhance agonist afﬁnities without affecting the magnitude of the maximal response. It seems, in fact, that the two GABAB receptor modulators, together with enhancers of the mGluR1 receptor, which were published at the same time (54), are the ﬁrst examples of allosteric drugs that not only enhance the potency, but also enhance the intrinsic efﬁcacy of agonists. In the meantime, several similar examples have been described for mGluR2 and mGluR4, as well as adenosine A3 receptors (55–58). This interesting observation raises the question of how the mechanism of action of these drugs functions. Radioligand-binding experiments provide a powerful tool to look deeper into the mechanisms underlying allosteric receptor modulation. The rate constants of association and dissociation of an orthosteric ligand are very sensitive to changes in receptor conformation induced by an allosteric agent acting at a distant site. Somewhat surprisingly, GS39783 was found to reduce the rate of association of the agonist radioligand [3H]3-aminopropylphosphinic acid ([3H]APPA) to native GABAB receptors from rat brain cortex, but this effect was overcompensated by an even greater reduction of the rate of dissociation, resulting in a net increase in afﬁnity (50). In fact, it is the balance of the changes in kinetic association and/or dissociation rate constants that makes up the effects of allosteric modulators on orthosteric ligand afﬁnity at binding equilibrium. Saturation curves with [3H]APPA to GABAB receptors in rat cortical membranes revealed an increase of the afﬁnity of the agonist radioligand in the presence of CGP7930; at the same time, a small, statistically not signiﬁcant increase in the maximal binding capacity was also observed (49). An increase of agonist afﬁnity produced by allosteric modulators was also visible in displacement experiments. The curves describing the inhibition of the binding of the antagonist radioligand [3H]CGP62349 to native GABAB receptors by agonists were biphasic, consisting of a high-afﬁnity and a lowafﬁnity component (50,51). GABAB receptors, like most GPCRs, have different agonist afﬁnities in their G-protein–coupled and –uncoupled states, which are most likely reﬂected in the two components of agonist displacement curves. Interestingly, the agonist afﬁnities of both components were enhanced by the allosteric modulators CGP7930 and GS39783. Moreover, the two modulators also increased the relative proportion of high agonist afﬁnity sites. This ﬁnding strongly suggests that the allosteric agents not only promote agonist binding to the orthosteric site, but also promote the

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interaction of GABAB receptors with their associated G-proteins. This mechanism might well contribute to the increase in maximal agonist stimulation observed in our functional assay systems. The increase in receptor–G-protein coupling also explains the small increase in the number of [3H]APPA-binding sites produced by CGP7930, the relevance of which was originally overlooked due to its lack of statistical signiﬁcance. This agonist radioligand only labels the high agonist afﬁnity (G-protein–coupled) receptor state, the proportion of which increases in the presence of positive modulators. On the other hand, the antagonist radioligand [3H]CGP62349 labels both receptor forms, which become visible in agonist-displacement experiments (see above). The maximal binding capacity for this radioligand remained unchanged in the presence of CGP7930 or GS39783, thus conﬁrming our original interpretation that the modulators do not act by unmasking additional GABAB receptors (59). Rather complex results, yielding further important insights into the mode of action of CGP7930, were obtained from radioligand-binding experiments performed with recombinant GABAB receptor preparations (49). In membranes from CHO cells expressing the GABAB(1) subunit only, the binding of the radioligand [3H]CGP62349 was not inhibited at all by CGP7930, conﬁrming the notion that this compound does not bind to the orthosteric receptor site. On the other hand, GABA displaced the binding of [3H]CGP62349 with low afﬁnity [inhibitory concentration 50 (IC50) ca 100 mM]. In this situation, the binding of GABA was not amenable to allosteric modulation; in fact, its afﬁnity remained unchanged in the presence of CGP7930. On the other hand, when the same experiments were performed using membranes from cells expressing both GABAB receptor subunits, biphasic displacement curves were obtained with GABA. The IC50 values of the low-afﬁnity component were close to those found with the GABAB(1) monomer and remained unchanged in the presence of CGP7930, whereas the IC50 of the high-afﬁnity component was decreased by CGP7930 from 5.1 (control) to 1.9 mM. The low-afﬁnity component comprised about 70% of the total ligand binding and was attributed to an overexpression of the GABAB(1) subunit in our stable cell line. Because the high-afﬁnity dimeric receptor form represented only a minor proportion of the total population of binding sites, it was not possible to recognize the expected further distinction between Gprotein–coupled and –uncoupled states within this component. These results demonstrate that the presence of the GABAB2 subunit is essential for the modulation of agonist-binding afﬁnity by CGP7930. They indicate that either the binding site for allosteric modulators is physically located on the GABAB2 protein or else this subunit is involved in another crucial fashion in the mechanism of modulation. Pin et al. (44,60) have proposed a model suggesting that allosteric modulators would interact with the seven transmembrane domains of either GABAB subunit. These predictions have now been conﬁrmed by the ﬁnding that CGP7930 directly activates the heptahelical domain of

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GABAB2, resulting in partial agonism in a highly sensitive assay system with a considerable degree of receptor reserve (61). Recent theoretical receptor models allow for allosteric modulators having some intrinsic efﬁcacy of their own via the allosteric-binding site (62). Thus, the ﬁndings of Binet et al. (61) are not at variance with the concept that CGP7930 is an allosteric GABAB receptor modulator. THEORETICAL ASPECTS OF ALLOSTERIC MODULATION; EFFECTS OF MODULATORS ON ORTHOSTERIC LIGANDS WITH DISTINCT INTRINSIC EFFICACIES The newly observed phenomenon that allosteric modulators in certain cases enhance not only agonist afﬁnity, but also efﬁcacy, is adequately described by recently developed theoretical receptor models (46,62). The model proposed by Hall (62) combines the previously known ‘‘two-state model’’ of receptor activation (63) with the ‘‘ternary complex model’’ for allosteric effects on binding afﬁnities (64), and at the same time introduces a novel activation cooperativity constant by which the intrinsic efﬁcacy of an orthosteric ligand is modiﬁed. This model treats allosteric interactions in neutral terms, that is, independently of the intrinsic properties of orthosteric ligands. From this it follows that partial agonists, inverse agonists, and competitive antagonists should a priori be as much amenable to allosteric modulation as full agonists. In fact, we have recently found that the binding afﬁnities of a number of competitive GABAB receptor antagonists were decreased in the presence of CGP7930 or GS39783 (59). Moreover, in the same study it was also shown that in a GTP(c)35S assay the intrinsic efﬁcacy of the partial agonist CGP47656 was enhanced by about fourfold by the two allosteric modulators. Previously, only about 1.5- to twofold increases were observed with GABA. Thus, apparently the less efﬁcacious receptor activation with a partial agonist leaves more room for positive modulation. Most interestingly, two compounds (CGP35348 and 2-hydroxy-saclofen), which have previously been considered as being neutral GABAB antagonists (2) and, accordingly, did not stimulate GTP(c)35S binding at all on their own, became partial agonists in the presence of CGP7930 or GS39783. Thus, either the modulators were able to confer partial agonistic activity to compounds otherwise devoid of any intrinsic efﬁcacy, or they ampliﬁed hidden, marginal agonistic properties of these two orthosteric ligands, which could normally not be detected in the apparently not sufﬁciently sensitive GTP(c)35S assay. The answer to this question was found using another experimental approach. GABAB RECEPTOR MODULATION IN CELLULAR AND PHYSIOLOGICAL ASSAY SYSTEMS Although experimental techniques using membrane preparations with native or recombinant receptors allow the detection of allosteric modulation

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and its molecular characterization, they represent a simpliﬁed, artiﬁcial system. It is therefore important to demonstrate that the pharmacological characteristics of allosteric modulation found in these assay systems are also valid in a more physiological context, that is, in cellular or intact tissue preparations. We have shown that CGP7930 and GS39783 enhance the GABAB receptor–mediated inhibition of adenylyl cyclase activity induced by a water-soluble forskolin analog in intact CHO cells (59). This experimental system turned out to be highly sensitive, the potency of GABA being considerably higher than that previously observed in GTP(c)35S assays, suggesting the existence of a signiﬁcant number of spare receptors. In line with this interpretation, a modest inhibition of cAMP formation produced by CGP7930 and GS39783 on their own was observed, indicating for the ﬁrst time in our experiments a low degree of partial agonistic activity, via the allosteric site, of the two modulators. A similar observation was also made by Binet et al. (61), equally using an assay system with a high degree of receptor reserve. This high sensitivity of the cAMP assay in our CHO cell line stably expressing the GABAB receptor also enabled us to demonstrate that CGP35348 and 2-hydroxy-saclofen do in fact have some weak intrinsic agonist efﬁcacy, which was enhanced by CGP7930 and GS39783, thus conﬁrming our previous results obtained in the GTP(c)35S assay. We have characterized the effects of CGP7930 and GS39783 on GABAB receptor function in various other cellular systems. In Xenopus laevis oocytes injected with messenger RNA (mRNA) for the two GABAB receptor subunits and for inwardly rectifying (Kir3) potassium channels, CGP7930 and GS39783 enhanced the stimulation of potassium currents by GABA (49,50). In the case of the former, these experiments were performed with both GABAB(1a/2) and the GABAB(1b/2) subunit combinations, with very similar results (49). No potassium currents were elicited in these oocytes in the presence of either of the modulators alone. In transiently transfected cell lines, GABAB receptors couple to the phospholipase C pathway if they are coexpressed with an appropriate chimeric G-protein (65). In such experiments in human embryonic kidney (HEK293) cells using a ﬂuorescence imaging plate reader, GABAB receptor–mediated intracellular calcium mobilization was enhanced by CGP7930 and GS39783 (49,50). As in the oocyte experiments with CGP7930, similar effects were produced by GS39783 with both GABAB(1) isoforms (50). Again, no intrinsic agonistic activity was observed with both modulators. Dissociated rat cortical neurons in primary culture form synaptically connected networks. In such a system, removal of Mg2þ from the incubation medium elicits synchronized intracellular calcium oscillations, resulting from the interplay of spontaneous depolarizations of inhibitory and excitatory neurons (66). The frequency of these oscillations was decreased by L-baclofen, and this effect was blocked by the competitive GABAB antagonist CGP54626A. CGP7930, at a concentration

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at which it had no effect on its own, further reduced the calcium oscillation frequency in the presence of L-baclofen (49). In a hippocampal slice preparation, the application of two consecutive electrical stimuli to afferent pathways results in inhibition of the second population response, due to the activation of local inhibitory GABAergic interneurons (67). Activation of presynaptic GABAB receptors results in a suppression of this ‘‘paired pulse inhibition,’’ probably through inhibition of GABA release from interneurons. Like baclofen, GS39783, when applied on its own, reversed paired pulse inhibition (50). The effects of both of the compounds were counteracted by the competitive antagonist CGP55845A. A competitive antagonist would not be expected to inhibit GABAB receptor activation by a compound acting at a site distinct from the orthosteric GABA-binding site. Therefore, we concluded that the effect of GS39783 on paired pulse inhibition was due to a potentiation of the activity of endogenous GABA, rather than to a direct activation of GABAB receptors (50). To summarize the available in vitro data, it can be said that in different experimental systems with increasing degrees of complexity, from molecular assays in membrane preparations to physiological readouts in neuronal networks in culture or intact tissue slices, CGP7930 and GS39783 act as allosteric enhancers of GABAB receptor–mediated responses. They increase the afﬁnities and efﬁcacies of GABAB agonists by (at least in the case of CGP7930) acting at the seven transmembrane domains of the GABAB2 subunit. The direct activation of GABAB receptors by these agents is negligible and becomes visible only in highly sensitive assay systems with a high degree of receptor reserve. ENHANCEMENT OF GABAB RECEPTOR FUNCTION BY OTHER MECHANISMS AND OTHER AGENTS The GABAB receptor shares sequence similarity with other members of Family 3 GPCRs, such as the extracellular Ca2þ-sensing receptors and mGluRs. Both of these are sensitive to Ca2þ: while the former is activated by calcium ions, certain mGluR subtypes have in fact been shown to be modulated by Ca2þ (68,69). On the other hand, the extracellular Ca2þ-sensing receptor is allosterically modulated by amino acids, in particular glutamate (70). In the light of these relationships, it is not surprising that the GABAB receptor is also sensitive to regulation by Ca2þ ions. In two independent studies (32,71) it was found that Ca2þ, at micromolar concentrations, enhanced the potency, but not the maximal effect, of GABA at native and recombinant GABAB receptors in GTP(c)35S experiments. Similar effects were found in adenylyl cyclase and potassium channel assays (32). With the exception of the cAMP assay, activation of GABAB receptors by baclofen was not at all affected by Ca2þ. Moreover, Ca2þ was found to enhance the binding afﬁnities for GABA, but not baclofen, for native and recombinant

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GABAB receptors (71). Unlike CGP7930, calcium also increased the binding afﬁnity of GABA in membranes from CHO cells expressing the GABAB(1) subunit only. Mutagenesis experiments identiﬁed serine(ser)269 in the GABAB(1) subunit as an amino acid, which is critical for the effect of calcium (71). The fact that Ser269 is located in the close vicinity of the GABA-binding site (19,21,22) raises the question of how calcium exerts its effect. An allosteric mechanism would imply a conformational change of the receptor protein, thus changing the positioning of Ser269 to optimize GABA binding (possibly by making the hydroxyl group of serine available for hydrogen bonding), without a direct physical interaction of Ca2þ with Ser269 and GABA. Alternatively, the proximity of Ser269 to the GABA-binding site strongly suggests that Ca2þ might enhance the binding afﬁnity of GABA by the formation of a complex, with one or two amino acid residues (Ser269, possibly also Tyr366) and GABA being bound as ligands around Ca2þ as the central atom (72). This second hypothesis might also explain the lack of calcium sensitivity of baclofen, which docks into the GABAB receptor cavity in a manner somewhat different from that of GABA. In addition to hydrogen bond and ionic interactions of its carboxylic acid and amino groups with the same receptor amino acid residues as with GABA, its binding seems to be stabilized by its p-chlorophenyl aromatic ring being stacked in a sandwich-like manner between two tyrosine residues (p–p interactions) (72). Although the two proposed mechanisms of action of calcium are clearly distinct, the seemingly more likely of them not being of allosteric nature, they appear to be difﬁcult to distinguish experimentally. Unlike the effects which Ca2þ has at calcium-sensing and mGluRs, which require millimolar calcium concentrations, the enhancement of GABA afﬁnity by Ca2þ has an EC50-value of 37 mM (71). Calcium concentrations in the cerebrospinal ﬂuid (and presumably also in the synaptic cleft) are in the millimolar range. Therefore, the physiological relevance of calcium effects at GABAB receptors is uncertain, although synaptic calcium concentrations might drop considerably in certain pathological situations, such as epileptic seizures (73,74) or ischemia (75). Under normal conditions, however, the calcium site in the GABA-binding pocket is most likely saturated, leaving no room for a regulatory function. Our early experiments with CGP7930 and GS39783 had all been carried out in the presence of maximally active Ca2þ concentrations; however, Olianas et al. (53) have recently shown that CGP7930 also modulates GABAB receptor function in the absence of calcium, although the effect on agonist potency (but not that on efﬁcacy) was smaller than in its presence. The extracellular Ca2þ-sensing receptor is allosterically modulated by amino acids and arylalkylamine-like molecules (70,76,77). This has prompted Kerr et al. (78,79) to examine the arylalkylamines fendiline, prenylamine, and ‘‘F551’’ as well as several amino acids and dipeptides for their actions on GABAB receptor–mediated responses. They found that baclofen-induced

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ﬁeld potentials in rat neocortical slices were enhanced by these agents. Because the compounds were without effect when applied alone, the authors strongly suggested that they would act as allosteric modulators at GABAB receptors. However, molecular aspects of the mode of action of these compounds were not addressed in the studies by Kerr et al. (78,79). The kind of enhancing effects observed by these authors in brain tissue slices might also well be due to, for example, receptor cross-talk or interneuron signaling (downstream effects), without the involvement of true allosteric mechanisms at the molecular level. We have completely failed to see any enhancement of GABA afﬁnity or efﬁcacy when we tested arylalkylamines, amino acids, and dipeptides at native and recombinant GABAB receptors in different biochemical assay systems (51). Olianas et al. (53) also failed to see an enhancing effect of fendiline in a GTP(c)35S assay, but in their hands it counteracted the allosteric effect of CGP7930 on GABA efﬁcacy (but not potency). The mechanism of this interaction is not clear at present. More investigations are needed to shed light on the mechanism by which arylalkylamines and amino acids enhance GABAB receptor–mediated responses in brain slices (see also Chapter 11). EFFECTS OF ALLOSTERIC GABAB RECEPTOR MODULATORS IN VIVO We have recently published a microdialysis study showing for the ﬁrst time at a biochemical level that GS39783 potentiates GABAB receptor-mediated responses in vivo (79a). Orally applied, the allosteric modulator enhanced the inhibition of cylic AMP formation produced by a threshold concentration of baclofen in a dose-dependent manner while lacking effects on its own. Investigations on the role of GABAB receptors in behavioral processes have hitherto mostly relied on the use of the prototype agonist baclofen. However, baclofen induces sedation, hypothermia, and muscle relaxation, which may signiﬁcantly interfere with behavioral measurements in experimental animals. This has increased the interest in studying allosteric modulators, which are potentially devoid of these side effects, in behavioral paradigms. The effectiveness of CGP7930 in vivo has been demonstrated by its ability to enhance the sedative/hypnotic effects (loss of righting reﬂex) of threshold doses of the GABAB receptor agonists baclofen and chydroxybutyrate (GHB) in DBA mice (80). On the other hand, in the dose range tested, CGP7930 did not induce any loss of righting reﬂex when administered alone. The established role of GABAergic neurotransmission in anxiety prompted Cryan et al. (81,82) to study the effects of GS39783 in various animal models for anxiety and depression. Acute and chronic treatment with GS39783 decreased anxiety in the light–dark box and elevated zero maze anxiolysis tests, but no effect of GS39783 was found in the forced swim test

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for antidepressant activity. The anxiolytic-like effects of GS39783 are in line with the ﬁnding that GABAB-knockout mice are more anxious than their wild-type littermates (81). The positive modulator did not have any of the side effects that are associated with baclofen and/or benzodiazepines, such as muscle relaxation, sedation, impairment of motor ability or cognitive function, and potentiation of the effects of ethanol (82). The rewarding effects of drugs of abuse are believed to be mediated through the activity of dopamine neurons in the mesolimbic system, projecting from the ventral tegmental area (VTA) to the nucleus accumbens (83). The activity of the dopamine neurons is under tonic inhibitory control by GABAergic interneurons in the VTA. GABAB receptors are located on both types of neurons. Baclofen has been shown in several preclinical and clinical studies to reduce the intake of drugs, such as cocaine or nicotine (84–86). For the apparent paradox that GHB, which is, like baclofen, a GABAB agonist (87–89), is a drug of abuse itself (90), a possible explanation has been proposed (91). It is related to the differential coupling of GABAB receptors to GIRK channels on mesolimbic dopaminergic cells and GABAergic interneurons, thus resulting in distinct levels of activation by high-afﬁnity (such as baclofen) and low-afﬁnity (such as GHB) agonists. Baclofen would activate GABAB receptors on both types of neurons, resulting in a net inhibition of dopamine release, whereas GHB at doses typical for recreational use would inhibit only GABAergic interneurons, thereby producing an increase in dopamine release in the nucleus accumbens (91). In line with the established role of GABAB receptors in the reinforcing and sensitizing effects of cocaine (92,93), Smith et al. (94) have examined the effects of CGP7930 and GS39783 on cocaine intake in rats. They observed that both drugs reduced cocaine self-administration under various schedules of reinforcement. No sedation or motor impairment was seen. The effects were very similar to those found previously (95) with baclofen at doses that did not alter foodmaintained responding, suggesting that these drugs attenuate the reinforcing effects of cocaine in the absence of sedation or motor impairment. CGP7930 was clearly more effective at reducing cocaine intake than GS39783, which was probably due to differences in the pharmacokinetic properties of the two drugs. The hypothesis that the two modulators enhance the tonic GABAergic inhibition of mesolimbic dopamine neurons, thereby counteracting the stimulating effect of cocaine, is appealing, but not yet proven. The proof that the effects of CGP7930 and GS39783 observed in animals are actually brought about through positive allosteric GABAB receptor modulation in vivo is difﬁcult to produce because of the lack of adequate pharmacological tools allowing experiments leading to unequivocal conclusions. CGP7930 and GS39783 have not yet been tested in animals lacking functional GABAB receptors. Only the GABAB(1)/ knockout mice generated on the Balb/c genetic background are viable; other GABAB(1)deﬁcient mice die within one month after birth (96,97), thus precluding

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the behavioral testing of adult animals. Drug effects in surviving Balb/c GABAB(1)/ knockout mice would most likely be confounded by the phenotype of these animals, which is characterized by a decreased epileptic seizure threshold, hyperlocomotion, hyperalgesia, and memory impairment (97). Therefore, GABAB receptor-deﬁcient animals would be difﬁcult to use as controls to assess the involvement of allosteric GABAB receptor modulation in behavioral readouts in vivo. Competitive GABAB antagonists are also likely to shift control baseline values in behavioral experiments, which would make their effects in combination with positive modulators difﬁcult to interpret. Neutral antagonists at the allosteric GABAB site, that is, compounds that would block the effects of modulators without affecting those of orthosteric agonists, in much the same way as ﬂumazenil antagonizes the effects of benzodiazepines at the GABAA receptor, are not available at present, but would be very useful tools for future pharmacological studies. OUTLOOK: POSSIBLE THERAPEUTIC APPLICATIONS OF POSITIVE GABAB RECEPTOR MODULATORS AND FUTURE PROSPECTS Research on allosteric modulation of GABAB receptors is in its early stage, and many open questions remain to be answered with regard to the mechanisms of action of such drugs in vitro and in vivo. Although CGP7930 and GS39783 are useful compounds to investigate these mechanisms in vitro, their use as in vivo tools is hampered to some extent by their relatively low potency and rather moderate pharmacokinetic properties. Thus, the discovery and optimization of novel types of molecules acting as GABABreceptor modulators with high potency, selectivity, and brain permeability will be an important challenge for pharmacologists and medicinal chemists. These qualities in novel molecules will be prerequisites for their use as tools to further investigate the in vivo effects of GABAB modulators, and for the development of therapeutically useful drugs. The clinical indications for which a therapy with allosteric GABAB receptor modulators might be successful remain to be identiﬁed. Preclinical and clinical pharmacological evidence and results obtained with GABAB knockout animals point to a possible role for GABAB receptor enhancers in gastrointestinal disorders, anxiety, epilepsy, pain, and drug (especially cocaine and nicotine) abuse (8,81,82,85,86,94,96–100). Drug abuse seems to be a particularly attractive indication for treatment with positive allosteric GABAB receptor modulators, not only because the concept has already been validated in experimental animals (94), but also because of the molecular mechanisms that seem to be involved. Xi et al. (92) found that after repeated cocaine administration to rats, GABAB receptor function in the nucleus accumbens, assessed by the stimulation of GTP(c)35S binding by baclofen, was reduced, without a

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concomitant reduction in the level of GABAB(1) or GABAB(2) proteins. Similarly, Amantea et al. (101) have reported that on repeated administration of nicotine to rats, GABAB receptor density and afﬁnity were not altered in different brain regions. However, the level of G-protein–coupling to the receptor was reduced in the prefrontal cortex and the nucleus accumbens. It is exactly in such a situation that one would expect allosteric enhancers to be of clinical beneﬁt, because they precisely act by improving the efﬁciency of the coupling of the GABAB receptor to its G-proteins. The GABAB receptor agonist () -baclofen (Lioresal) has been in clinical use as an antispastic agent for several decades. However, the strong muscle-relaxant property of this drug also precludes its therapeutical application in other indications, in which it would be an unwanted side effect. On the other hand, the fact that allosteric modulators are, as should be expected on theoretical grounds, devoid of the side effects associated with the GABAB agonist baclofen in animal experiments means that spasticity will not be a potential indication for such compounds. Thus, it seems that GABAB receptor agonists and positive modulators might well cover complementary sets of clinical indications. This raises the hope that the pharmacological principle of positive allosteric GABAB receptor modulation will lead to a new generation of useful therapeutic agents.

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11 Allosteric Interactions at GABAB and Related G-Protein–Coupled Receptors David I. B. Kerr and Jennifer Ong Department of Anaesthesia and Intensive Care, The University of Adelaide, Adelaide, South Australia, Australia

INTRODUCTION Allosteric modulation of G-protein–coupled receptor (GPCR) function is now well recognized as an important feature of its pharmacology, particularly among receptors of Family 1 and Family 3. The notion of allosteric actions at proteins goes back many years, to studies on enzyme activity where product inhibition was ﬁrst seen (1). Allosteric means ‘‘other shape,’’ i.e., the allosteric ligand is not a steric analog (isosteric) that can ﬁt at the receptor ligand-binding site. At the molecular level, allosteric ligands are considered to combine at a site on the receptor molecule other than that occupied by the true orthosteric ligand. Occupation of the allosteric site induces an alteration of the receptor’s molecular structure, in such a way as to change the binding of the orthosteric ligand (2,3). However, this is by no means universally true, as changes in afﬁnity or efﬁcacy can occur (4). It is also axiomatic from thermodynamics that, just as allosteric binding inﬂuences orthosteric binding, the converse is also true, with orthosteric binding leading to changed binding of the allosteric ligand. Moreover, because agonist binding to the receptor changes the afﬁnity of the associated G-protein for the nucleotide, thermodynamically, then, nucleotide binding will in turn alter the afﬁnity of the receptor for its agonist ligand. These kinds of allosteric interactions are examples of the strongly maintained 259

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tenet, in molecular biology, that any allosterically induced conformational change at the receptor will alter the receptor afﬁnity for the agonist. In practice, such reciprocal allosteric effects on binding do not always occur, but these modulatory actions can still be discerned in functional assays (5). Here, we examine the implications on this seeming anomaly in the special case of c-aminobutyric acidB (GABAB) receptors within the context of Family 3 receptors. ORIGIN OF FAMILY 3 GPCRs Since the molecular evolution of receptor proteins and their respective genes is recorded in their nucleotide sequences, we have some insight into their genetic history (6). For example, a Family 3–like GPCR already appears in the sponge (Geodia sp.), with a mixed metabotropic glutamate (mGlu)/GABAB-like heptahelical domain (7). It would be of great interest to know if this primordial (500 million years old) metazoan receptor is already capable of modulation by allosteric agents known to act at the heptahelical domain in mammals. Certainly, receptors with the pharmacological proﬁle of typical GABAB receptors are present in holothurians (8) and crayﬁsh (9). It is generally accepted that Family 3 GPCRs likely originated from an early gene fusion of a periplasmic amino acid binding protein, as found in gram-negative bacteria, with a primordial rhodopsin-like receptor, giving the GPCR Venus ﬂy trap (VFT) and heptahelical domains, respectively. Phylogenetic analysis of the gene structure for either the VFT region (10) or the heptahelical domain (11) reveals similar interrelationships across the Family 3 members (12,13). In this, GABAB receptors are nearest to the rhodopsin stem, with the calcium-sensing receptors next nearest, and associated with taste receptors (TAS1Rs) (14), as well as putative pheromone receptors for the vomeronasal organ (V2Rs) (15). The latter are homologous with the calcium-sensing receptors, particularly in possessing a VEV(D/E) peptide sequence at the junction of the external 03 loop with transmembrane 7 (TM7). This is a site for allosteric modulation in calcium-sensing receptors (16). Most distant from the ‘‘rhodopsin stem,’’ and showing a number of subtypes, are the metabotropic glutamate receptors (mGluRs), of which mGluR1 and mGluR5 are nearest to the calcium-sensing receptors, with mGluR7 and mGluR8 the most distant. It is noteworthy that the sequences of GABAB1 and GABAB2 subunits are sufﬁciently remote from the remainder of Family 3 that they can be considered as a separate group wherein the VFT is more closely linked to the heptahelical domain than in all others of Family 3. A heptahelical region coupled to G-proteins is the overall deﬁning feature of the GPCR superfamily, while differentiation within the superfamily is essentially based on the size and structure of the N-terminal domain.

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Members of the Family 1 amine-receptor cluster have relatively short N-terminals, whereas that of the Family 3 have a uniquely large, bilobed structure that is likened to a VFT, where the lobes of the N-terminal domain can open and close to bind orthosteric ligands, and so activate the receptor. In contrast, in GPCRs of Family 1, orthosteric ligands bind directly at the heptahelical region, as do allosteric agents, with binding involving a crevice formed by TMs 3, 5, 6, and 7. This region is also the binding site for the primordial opsin receptors (17). Elements of this groove character persist in Family 3 receptors, where most, if not all, allosteric modulators bind at the heptahelical domain (12,18). Both TM3 and TM6 move upon binding of the ligand, which leads to activation of the G-protein (12). The possibility of a relatively small ligand molecule binding across otherwise seemingly remote TMs, e.g., 3, 5, and 7, is immediately apparent, as these are relatively close within the groove. It is of particular interest to see this degree of resemblance in the molecular basis of modulation within these Family 1 and Family 3 GPCRs. Activation of the heptahelical domain leads to a release of the a,b,c-Gproteins to produce a GPCR response involving cytoplasmic loops i2 and i3. One of the more curious aspects of the GABAB receptor heterodimer is that this feature occurs only with GABAB2 (19), although GABAB1 bears the ligand recognition site (20,21). Importantly, a cyclic change between guanosine 50 -triphosphate (GTP) and guanosine 50 -diphosphate (GDP) during activation leads to an allosteric interaction with the receptor–ligand afﬁnity, where the GTP-occupied complex has a lower agonist afﬁnity than the GDP form. This change in afﬁnity with GTP was ﬁrst shown for the GABAB receptor by Bowery et al. (22), and provided the original clue that these receptors belong to the GPCR superfamily. Such a notion was conﬁrmed by Andrade et al. (23), who showed that the more stable GTPbS, which cannot yield GDP, would abolish baclofen activation of the GABAB receptor. Subsequently, we showed that GABAB receptor–mediated responses to baclofen are blocked by amiloride (24); this action is now considered to be due to negative allosteric modulation of G-protein coupling at the receptor (25). Such interactions between receptors and G-proteins are primary allosteric effects, but other allosteric modulatory actions are known for various members of Family 3 GPCRs. ALLOSTERIC MODULATORS FOR FAMILY 3 GPCRs There are both positive and negative allosteric modulators for Family 3 GPCRs. Curiously enough, the ﬁrst modulators were identiﬁed before the existence of this family of receptors was realized, following the discovery of the calcium-sensing receptors that control intracellular calcium levels via Gq11, phospholipase C and inositol trisphosphate (26). It was already known that certain phenylalkylamine derivatives could alter intracellular

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calcium levels, by an action unrelated to activation of calcium channels. Systematic structure–action studies of this effect yielded a considerable group of the so-called ‘‘calcimimetic phenylalkylamines’’ (27), now known to be allosteric modulators (28) for calcium-sensing receptors. MODULATORS AT mGluRs At present, among Family 3 receptors, the widest range of chemically discrete allosteric modulators is found for mGluRs, where some 20 different chemical classes of modulator exist. The ﬁrst of these were noncompetitive antagonists at mGluRs, which uniformly fail to inﬂuence agonist binding at these receptors. Their modulatory sites are located at, or near, the extracellular surface of TM7, TM6, and TM3 of the heptahelical domain, analogous to the opsin-binding groove of Family 1 receptors (29). This binding evidently disrupts mGluR1 activation by inhibiting the interaction between the agonist-bound VFT and the heptahelical domains. The original noncompetitive antagonists (Fig. 1) were 2-quinoxaline-carboxamide-N-adamantan-1-yl (NPS 2390) (30), and () 7-(hydroxyimino) cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), active at mGluR1 (31,32), as well as 6-methyl-2-(phenylazo)-pyridinol (SIB-1757)

Figure 1 Representative examples of the chemical structures of a variety of allosteric modulators for metabotropic glutamate receptors. All of these compounds modulate their receptors by binding at their heptahelical domains, without inﬂuencing agonist binding.

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and 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP), active at mGluR5 (33,34). Some notion of the variety of noncompetitive negative allosteric modulators, for the Group I mGluRs, can be gained from the works of Malherbe et al. (35) and Lavreysen et al. (29). Moreover, there is also a whole family of noncompetitive mGluR1 antagonists, based on 2,4dicarboxy-pyrroles (36). Most interestingly, O’Brien et al. (37) recently reported a series of benzaldazine analogs whose activities vary from negative modulators [such as 3,30 -dimethoxybenzaldazine (DMB)], to positive modulators [such as 3,30 -diﬂuorobenzaldazine (DFB)] at the mGluR5 subtype. None of these benzaldazines, positive or negative modulators, affect binding of the agonist [3H]quisqualate. But they do compete with [3H]-3-methoxy-5(2-pyridinylethenyl) pyridine ([3H]methoxyPEPy), a noncompetitive antagonist acting at the TM7 binding region (37). Evidently, many noncompetitive antagonists at mGluRs, such as (3aS, 6aS)-6a-naphtalen-2-ylmethyl-5-methyliden-hexahydro-cyclopenta[c]furan1-on (BAY 36–7620) (Fig. 1), are in fact inverse agonists that nevertheless modulate at these receptors (38), while an apparently competitive antagonist 4-carboxy,3-hydroxy-phenylglycine (4-CHPG) at mGluRs is revealed as a partial agonist in the presence of the potentiating diﬂuorobenzaldazine (37). Moreover, ()-N-phenyl-7-(hydroxylimino)-cyclopropa[b]-chromen1a-carboxamide [()-PHCCC] (an analog of the mGluR1 negative modulator, CPCCOEt) is a weak negative modulator at mGluR1s, but is an effective positive modulator of mGluR4 (39). In the same way, 2-methyl-6-(2-phenylethenyl) pyridine (SIB-1893) and MPEP (analogs of the mGluR5 negative modulator SIB-1757; Fig. 1) are also positive modulators of mGluR4 (40). With these, at least MPEP has no effect on binding of the mGluR4 agonist L-(þ)-2-amino-4-phosphonobutyric acid (L-AP4). All such modulators bind at the heptahelical domain (29,34,41,42), as do the allosteric compounds such as N-(4-)2-methoxyphenoxy-phenyl-N-(2,2,2triﬂuoroethylsulfonyl) pyridylmethylamine (LY487379), and N-(4-phenoxyphenyl)-(3-pyridinylmethyl) ethanesulfonamide (LY181837) (Fig. 1), together with several analogs (43) that are both partial agonists and positive modulators at the mGluR2. All mGluR noncompetitive antagonists thus bind, and compete at a common region on the heptahelical domain, as do a number of positive modulators. Furthermore, LY487379 provides an example of a positive modulator that can convert partial agonists to full agonists at mGluR2s [Schaffhauser et al. (44)] (Fig. 2). This action, together with the conversion of a competitive antagonist to a partial agonist mentioned above, illustrates that positive allosteric modulators can alter efﬁcacy, presumably by changing the coupling efﬁciency or cooperativity at the G-protein without necessarily changing the agonist-binding afﬁnity. All of these modulatory effects, together with G-protein contributions, can be effectively modeled (5,45–47).

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Figure 2 Positive modulators at GABAB receptors, showing the relationship between calcimimetic compounds and CGP antagonists. Naphthyl-459 is a weak potentiator at GABAB receptors that exhibits considerable potency at neuronal calcium-sensing receptors, while 3-Cl,4-OMe-FEND is a highly potent potentiator at GABAB receptors that shows little or no activity at neuronal calcium-sensing receptors. CGP 7930 and GS 39783 are the original GABAB receptor allosteric modulators. Abbreviations: GABAB, c-aminobutyric acidB; 3-Cl,4-OMe-FEND, 3-Cl, 4-OMe-fendiline.

CALCIUM-SENSING RECEPTORS Phenylalkylamine Modulators Both positive and negative allosteric modulators also exist for the calciumsensing receptors, based on an arylalkylamine structure. The original positive modulators were (R)-N-(3-methoxy-2-phenylethyl)-2-(20 -chlorophenyl -1-propylamine) [(R)-NPS 467] and (R)-N-(3-methoxy-a-phenylethyl)-2(20 -chlorophenyl-1-propylamine) [(R)-NPS 568]. A family of aryl-sulfonylphenylpropane-diamines has also been described (48). But all these have been superseded by (R)-()-a-methyl-N-(3,3-(triﬂuoromethyl)propyl)-1-naphthalenemethanamine [(R)-AMG073] (Cinacalcet) with an improved metabolic proﬁle (49), and by (R)-2-[1-[1-naphthyl]ethylaminomethyl]-1H-indole (Calindol) (50). These compounds are known as calcimimetics, but normally still require the presence of calcium for their action. There are fewer negative modulators for calcium-sensing receptors, but N-(R)-2-hydroxy-3-(2-cyano3-chlorophenoxy)propyl-1,1-dimethyl-2-(2-naphthyl)ethylamine (NPS 2143) (51) and the somewhat related (1S,2S,10 R)-N1-(4-chlorobenzoyl)-N2-[1-[1naphthyl]ethyl]-1,2-diaminocyclohexane (Calhex 231) (52,53) are known.

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The availability of these new positive and negative modulators led to modeling of their binding sites at the calcium-sensing receptor (52–54). In this, a pivotal role of the glutamate residue E837 at or near the top of TM7 of the heptahelical domain is generally recognized, since this acidic residue provides ionic binding with the amine nitrogen of the phenylalkylamines. In addition, there are two hydrophobic pockets that accept the benzyl or naphthyl ring of the N-a-benzyl moiety and the aromatic terminal of the phenylpropyl moiety, respectively. It was eventually established that these calcimimetic effects at calciumsensing receptors are, in fact, due to allosteric modulation of the actions of extracellular calcium ions at these receptors (28). It has never been established by ligand-binding studies that these mimetic compounds are true allosteric modulators at calcium-sensing receptors; indeed, it might be quite difﬁcult to do so. Nevertheless, the results from molecular biology and expression systems establish beyond doubt that calcium binds at the VFT, whereas the phenylalkylamines bind at the heptahelical domain of the calcium-sensing receptor, clearly indicative of allosteric modulation (16,55). It was the striking structural resemblance between these phenylalkylamines and some ligands for GABAB receptors (56,57) that originally prompted us to examine their activity at the latter. Indeed, the phenylalkylamine structures differ only in the substitution of an aromatic for the phosphinic moiety in the GABAB receptor antagonists (Fig. 2). Amino Acid Modulators at Calcium-Sensing Receptors Some L-amino acids positively modulate calcium-sensing receptors (58). The mode of action of these amino acids at the receptor is unusual, in that they bind at the VFT terminal domain where their binding site overlaps that for calcium itself. Very high millimolar concentrations of the active aromatic amino acids are required for potentiation to occur, and some (e.g., branched chain amino acids) are virtually inactive. It is clear that the amino acids act at a different site from that for the phenylalkylamines (59), the actions of the phenylalkylamines being less dependent on calcium binding. In fact, they still raise the intracellular calcium levels in the absence of calcium binding at the VFT, or even of the VFT itself. The phenylalkylamines thus appear to be examples of agonists that bind at the heptahelical domain, and not the VFT of this Family 3 GPCR, and yet are also positive modulators. Calcium-sensing receptor activation by calcium induces intracellular calcium oscillations (60), superimposed on a raised background of internal calcium. At appropriate low levels of intracellular calcium, allosteric activation of the receptor by L-amino acids also elicits calcium oscillations, but these are of lower frequency and return to the baseline, i.e., without a raised background level (61). Furthermore, in the presence of phenylalkylamine allosteric modulators, intracellular calcium oscillations are induced

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at lower external calcium levels; this effect is still seen for a relatively prolonged period, following washout of a brief phenylalkylamine modulator treatment prior to altering the external calcium level (60). Such calcium oscillations are a rather characteristic feature of responses to submaximal agonist activation at GPCRs coupled to internal calcium (61). With the calcium-sensing receptor, they also provide an indication of the extent to which an allosteric modulator is affecting such coupling, which is of importance when considering this receptor in relation to allosteric modulators of GABAB receptors. ALLOSTERIC MODULATION AT GABAB RECEPTORS Recently, a number of reviews have been published on GABAB receptor function and molecular biology (13,62–68). By their nature, the GABAB receptor heterodimers, GABAB1/GABAB2, exhibit a high order of allosteric modulation in their function. GABAB2 normally modulates ligand binding at GABAB1, and it is remarkable that although GABAB2 apparently does not bind any ligand, GABAB2 is modulated when GABAB1 binds with an agonist (21). As a result, the modulated GABAB2 activates its associated G-proteins to elicit a response. Although associated G-proteins are missing from GABAB1, nevertheless, there are descriptions of responses to GABAB1, in the absence of GABAB2 subunits (69,70); these results remain unexplored. What might be the result of modifying GABAB2 by substituting a VFT from GABAB1 for the VFT of GABAB2, so that both GABAB1 and GABAB2 subunits can be directly activated with GABAB receptor agonists? In this arrangement, GABA no longer acts as a full agonist but instead becomes a partial agonist/antagonist. Furthermore, the antagonist (þ)-(S)-5,5dimethylmorpholinyl-2-acetic acid (Sch 50911) becomes a partial agonist at this modiﬁed heteromer bearing two VFTs from the GABAB1 receptor (71). These unusual changes in ligand properties at the heteromer, resulting from altering the GABAB2 receptor, so that it too can bind a GABA ligand, have not received the attention one might expect. But they are of signiﬁcance in the present context, as they show the extent to which altered allosteric modulation can modify receptor properties (72). Importantly, the notion that no agonist normally exists for the native GABAB2 receptor has been emphasized (21,65), despite the fact that the VFT for GABAB2 has long persisted in evolution. Elements of primordial bacterial periplasmic amino acid–binding proteins (73–75) still survive in its sequence. Although the VFT sequence of GABAB2 lacks many features that are found in GABAB1, this does not entirely rule out the possibility that some related agonists, such as selected amino acids, might in fact be agonists at the GABAB2 receptor. Such broad tuning of receptors for amino acids is rather well established (76,77); a variety of amino acids modulate the calcium-sensing receptor (58), while the umami (delicious) taste receptor

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dimer, T1R1 and T1R3, which is strongly positively modulated by inosine monophosphate, also responds to a broad range of L-amino acids (78). Likewise, T1R2 and T1R3 sweet receptors respond to ‘‘sweetener’’ peptides such as N-L-aspartyl-L-phenylalanine-1-methyl ester (aspartame). Indeed, we have already shown that some of the coded amino acids and their peptides potentiate the action of baclofen at GABAB receptors in rat neocortical slices (79). CALCIUM POSITIVELY MODULATES GABAB AND mGlu RECEPTORS Inorganic modulators of GPCRs are well known, especially in Family 1 receptors where sodium is a negative modulator (4). In addition, calcium is of particular importance as a positive modulator for Family 3 receptors, including mGluRs and GABAB receptors. Indeed, calcium was the ﬁrst positive allosteric modulator to be identiﬁed for GABAB receptors, where it exhibits an effective concentration 50 (EC50) of 37 mM in potentiating GABA at these receptors (80,81). This concentration of calcium is well below that generally found in plasma and cerebrospinal ﬂuid, so that GABAB receptors presumably normally act in the fully calcium-modulated state. Interestingly, baclofen actions at GABAB receptors are not modulated by calcium; the signiﬁcance of this is not known beyond the fact that allosteric modulation at a given receptor is often dependent on the agonist. As with calcium-sensing receptors, the binding site for calcium as a modulator is also at the VFT of mGluRs and GABAB receptors. Surprisingly, in calcium-sensing receptors, the binding site for calcium overlaps with that for amino acid potentiators at these receptors (82). From the sequence alignment, the GABAB2 receptor contains more conserved residues for amino acid potentiator binding than does the GABAB1 receptor that lacks the a-amino binding moiety of these other receptors (82–84). It follows that, although the GABAB1 VFT is undoubtedly the binding site for GABA ligands (21), there is a strong possibility that GABAB2 VFT may bind a variety of amino acids, just as does the VFT of the calciumsensing receptors. If this is so, then the VFT of the GABAB2 receptor is a possible binding site for the amino acid positive modulators we have found for the GABAB receptor dimer (79). CGP 7930 AND GS 39783 ARE ALLOSTERIC MODULATORS AT GABAB RECEPTORS A propofol analog, 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)phenol (CGP 7930) (Fig. 2) is a positive allosteric modulator of GABAB receptors (85,86). This molecule is a curious hybrid of propofol and gamma-hydroxy-butyric acid (GHB). Of particular relevance in the present

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context, dose–response curves for GABA in the presence of CGP 7930 show an increase in both potency and efﬁcacy of GABA at the GABAB1/ GABAB2 receptor heterodimer, when coexpressed in Xenopus oocytes with potassium inward-rectifying (Kir3) channels. Most recently, the actions of CGP 7930 at GABAB receptors were reexamined by Binet et al. (87), who showed that this modulator is in fact a partial agonist that binds at the heptahelical domain of GABAB2, and can elicit a response in the absence of a GABAB receptor agonist. Interestingly, CGP 7930 does not even require the GABAB2 VFT for its action, but directly activates the receptor through the heptahelical domain. Thus, CGP 7930 may be considered as the ﬁrst agonist found for the GABAB2 subunit, although it binds at a unique site. This situation invites comparison with the ability of the heptahelical domain of mGluR5 to behave as a rhodopsin-like receptor (88), where the positive allosteric modulator DFB likewise still acts as a full agonist in the absence of the mGluR5 extracellular VFT, and therefore has no effect on agonist binding. In the same vein, several allosteric modulators of mGluR2s are also partial agonists that bind and act at the TM7 region of these receptors (43). Thus, it is entirely possible for allosteric modulators of Family 3 receptors to have no effect on agonist binding at the VFT domain. It has not escaped us that if CGP 7930 binds at the heptahelical domain of GABAB2, then there is also the possibility that the active positive modulatory amino acids might do likewise. The drug of abuse GHB, which has been shown to activate GABAB receptors (89), resembles a CGP 7930 analog. GHB speciﬁcally acts at G-protein–gated inwardly rectifying potassium (GIRK, Kir3) channels linked to GABAB receptors on GABAergic neurons, but not on dopaminergic neurons (90). This points to the possibility that GHB could be an allosteric modulator, as such discriminations are not found using a ‘‘typical’’ receptor agonist. This is reminiscent of another compound gabapentin that activates GABAB heteroreceptors but not autoreceptors (91), and thus could also be an allosteric modulator. Gabapentin is generally described as a GABA-related analog, but it is a spiro-compound that assumes a conformation where the acid and amine moieties mimic an a-amino acid. Because of this resemblance, the question of whether gabapentin also might be an allosteric modulator at GABAB receptors is worth pursuing. More recently, a further group of positive allosteric modulators at heteromeric GABAB receptors have been described, including NN0 dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS 39783) (Fig. 2) and related analogs (92). In Xenopus oocytes, coexpressing the GABAB1,2 receptor and Kir3 subunits, GS 39783 also gives an increase of agonist afﬁnity and efﬁcacy. Unfortunately, there is no information on the allosteric binding site for GS 39783, but in keeping with the binding site for the modulators at mGluRs, and CGP 7930 at the GABAB2 subunit, one might suggest that it is also likely to be at the heptahelical domain of the latter subunit.

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PROPOSED SITE OF ACTION OF ARYLALKYLAMINES AT GABAB RECEPTORS A range of phenylalkylamine analogs of fendiline such as N-(3,3-diphenylpropyl)-a-methyl-3-methoxybenzylamine (F 551), (R,S)-467, (R)-N-(3-phenylpropyl)-a-methylbenzylamine [(R)-459], and the naphthyl analog of 459, (R)-naphthyl-459 (Fig. 2) are allosteric modulators at calcium-sensing receptors. These compounds are also potentiators of baclofen responses mediated through GABAB receptors in rat neocortex (56,57). We propose that such arylalkylamines might act at the heptahelical region of GABAB receptors to potentiate GABAB receptor–mediated responses in rat neocortex (56,57). In the GABAB1a,b subunits, an aspartate at the top of TM7 (D830 and D714, respectively) replaces the glutamate (E837) found at that location in calcium-sensing receptors, whereas in GABAB2, this aspartate (D714) is displaced by three residues along the 03 external loop, more toward TM6. At any event, there is an acidic functionality at GABAB receptors, close to the appropriate region of the heptahelical domain, where the basic amino group of the modulatory phenylalkylamines might bind at one of the subunits of the GABAB receptors; furthermore, there are adjacent phenylalanines (F832, 765, and F720) that could provide p-bonding of the a-methylbenzyl moiety, just as that which occurs in the calcium-sensing receptor. We do not yet have a negative modulator for GABAB receptors, in the phenylalkylamine series, although the negative modulators for calciumsensing receptors (51,52) suggest that such negative modulators for GABAB receptors might also exist. Contrary to Urwyler et al. (93), we maintain that the sum total of our various ﬁndings is entirely compatible with the phenylalkylamines acting as allosteric modulators of the GABAB1/2 receptor heterodimer. These phenylalkylamines may be partial agonists at the GABAB receptor heptahelical domain, exerting no action on agonist binding, as found in many other Family 3 receptors. Apart from the previously reported phenylalkylamine derivatives that potentiate GABAB receptor–mediated actions (56,57), we have found a new structurally related potentiator, (þ)-N-1-(3-chloro-4-methoxyphenyl)ethyl3,3-diphenylpropylamine 3-chloro,4-methoxyfendiline [(3-Cl,4-OMe-fendiline) (Fig. 2)] which is some 100 times more potent than the earlier analog 3-methoxy-fendiline, F 551 (94). In slices preincubated with either [3H]GABA or [3H]glutamic acid, 3-Cl,4-OMe-fendiline (1 mM) potentiated the inhibitory effect of baclofen (2 mM) on the electrically evoked release of [3H]GABA, and had a similar potentiating effect on the inhibitory effect of baclofen on the release of [3H]glutamic acid at a concentration of 0.5 mM, without affecting the basal release (94). 3-Cl,4-OMe-fendiline is thus a relatively potent potentiator at GABAB presynaptic hetero- and autoreceptors. Our above ﬁndings suggest that 3-Cl,4-OMe-fendiline is a potent potentiator of both pre- and postsynaptic GABAB receptor–mediated functions.

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Since these pre- and postsynaptic receptor activities differ in their effector mechanisms, it is unlikely that such enhancing actions induced by 3-Cl,4OMe-fendiline on GABAB receptors are mediated through downstream signaling cascades, as these differ for the two receptor subtypes. Rather, the potentiating effects of 3-Cl,4-OMe-fendiline on these receptors are more likely to arise from an increase in G-protein coupling, due to an action at the 7-transmembrane region. Interestingly, 3-Cl,4-OMe-fendiline, although being a highly potent modulator at GABAB receptors, does not produce signiﬁcant neuronal calcium oscillations even though it is presumably a calcium-sensing receptor modulator. In contrast, (R)-naphthyl-459 not only potentiates baclofen actions but also induces undesirable, marked calcium oscillations due to coactivation of neuronal calcium-sensing receptors in neocortical slice preparations (57,95). Therefore, 3-Cl,4-OMe-fendiline appears to be more selective for the modulatory site on GABAB receptors than for that on the calcium-sensing receptors. L-AMINO

ACIDS POTENTIATE BACLOFEN RESPONSES IN RAT NEOCORTICAL SLICES Superfusion with the GABAB receptor agonist baclofen, over a concentration range of 3–200 mM, consistently induces concentration-dependent hyperpolarizing responses in rat neocortical slices. These population hyperpolarizations are induced by GABAB receptor agonists and are sensitive to GABAB receptor antagonists. They are mediated through activation of inwardly rectifying potassium channels, blocked by barium (0.1 mM) or cesium (1 mM) (96). From our earlier studies, not only phenylalkylamines but also selected neutral L-a-amino acids and their dipeptides reversibly enhance GABAB receptor–mediated hyperpolarizing responses to baclofen (56,57,79). For example, the more potent amino acids such as L-Leu, L-Ileu, and L-Phe are equipotent in potentiating baclofen-induced responses (with an estimated EC50 of 50 mM for each amino acid). A complex hyperpolarization is typically seen with baclofen when combined with a potentiating amino acid, as shown previously (79); an initial spike with a rapid onset is a salient feature of these responses, often overshooting the subsequent slower plateau component. Furthermore, both L-Leu (100 mM) and the GABAB receptor positive modulator CGP 7930 (10 mM) produced such identical responses in the presence of baclofen [Kerr and Ong (79) (Fig. 1)]. The resemblance between potentiation at GABAB receptors with CGP 7930, and that seen with amino acids, is so striking as to suggest that these all act at some common region on the GABAB2 subunit. These effects are of particular interest in the light of the recent demonstration that CGP 7930 is in fact a partial agonist, acting at the heptahelical domain of the GABAB2 receptor (87). As far as allosteric interactions are concerned, the implication

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is that, by acting at GABAB1, baclofen positively modulates the action of the partial agonist CGP 7930 on GABAB2 which itself in turn modulates GABAB1. We propose that some amino acids, when applied together with baclofen, produce a fully potentiated hyperpolarizing response at the heterodimer, by acting at the GABAB2 receptor.

POSITIVE ALLOSTERIC ACTIONS OF AMINO ACIDS AT RECOMBINANT GABAB RECEPTORS As an example of allosteric modulation of GABAB receptors by amino acids, we have examined the modulatory effect of L-Phe on recombinant GABAB receptors (GABAB1a and GABAB2), coupled to Kir3 channels expressed in Xenopus laevis oocytes. This was done in collaboration with Dr. Mary Collins (University of Sydney). In such isolated expression systems, there can be no downstream effects generated by modulators coapplied with agonists, since oocytes lack the necessary proteins. Using a two-electrode voltage clamp, L-Phe (300 mM) on its own did not activate the channels, but in combination with a submaximal concentration of GABA (3 mM) potentiated the GABA-induced hyperpolarization by almost twofold (Fig. 3) (Collins, Kerr, and Ong, unpublished observations). Similar results were obtained using baclofen as the agonist. In oocytes requiring high concentrations of amino acids to elicit a modulation of the GABAB1/2 receptor response, maximal potentiation requires up to 1 mM of amino acid, while in others, the EC50 for potentiation is less than 10 mM, reﬂecting variations in the expression products. Contrary to Urwyler et al. (93), these results show that such amino acids can be true allosteric modulators at recombinant GABAB receptors.

L-Gln, L-Asn

AND L-Orn ARE ALSO POTENT POSITIVE MODULATORS OF GABAB RECEPTORS Originally, we employed the same L-amino acids as those used on the calcium-sensing receptors (58) to look for any effects on GABAB receptors. Many of these positively modulate GABAB receptor–mediated actions, but with potency proﬁles quite different from those seen at calcium-sensing receptors (79). For instance, histidine is highly active as a potentiator at calcium-sensing receptors, but is inactive at GABAB receptors. Conversely, the branched chain amino acids L-Leu and L-Ileu are rather effective potentiators at GABAB receptors but are virtually inactive at calcium-sensing receptors. Since L-glutamine (L-Gln), L-asparagine (L-Asn) and L-ornithine (L-Orn) are present in the cerebrospinal ﬂuid at appreciable levels (317.58, 3.65, and 2.51 mM, respectively) (97–99), we have now used them to see if they also modulate baclofen responses at GABAB receptors, in rat brain slices.

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Figure 3 Effects of GABA and L-Phe on recombinant GABAB1a/2 coupled to the voltage-gated potassium channels, GIRKs 1 and 4 expressed in Xenopus oocytes. Effects were recorded using two-electrode voltage clamp electrophysiology, and current responses were obtained in the presence of 45 mM Kþ buffer when the oocyte was clamped at 60 mV. GABA (30 mM; duration indicated by ﬁlled bar) produced a maximal response. L-Phe (300 mM; duration indicated by hatched bar) was without effect when applied alone. However, in the presence of a submaximal response to GABA (3 mM; duration indicated by open bar), L-Phe (50 mM; duration indicated by grey bar) enhanced the effect of GABA by 13%. The positive modulatory effect of L-Phe was dose dependent. At a higher concentration, L-Phe (300 mM) enhanced the GABA effect by 87%. Abbreviations: GABA, c-aminobutyric acid; GIRK, G-protein–gated inwardly rectifying potassium.

Each of these reversibly potentiated baclofen-induced hyperpolarizing responses in a concentration-dependent manner, in the low micromolar range. The agonist baclofen (10 mM) applied alone for three minutes induced a control hyperpolarizing response. After a 30-minute tissue washout, coapplication of L-Gln (0.3 mM) and baclofen (10 mM) for four minutes increased the response to baclofen in a signiﬁcant manner, giving a typical initial spike followed by a more prolonged recovery (Fig. 4), as seen with the original amino acids (79); likewise, L-Orn (1 mM) (Fig. 4) and L-Asn (5 mM) (ﬁgure not provided) induced similar effects. The EC50 values for these amino acids are 0.2 mM for L-Gln, 1 mM for L-Orn, and 10 mM for L-Asn. Moreover, the

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Figure 4 Discontinuous record of the hyperpolarizing effects of baclofen (10 mM) in a rat neocortical slice preparation, and the potentiating effects of L-Gln (0.3 mM), as well as L-Orn (1 mM) on baclofen-induced responses. The control response to baclofen was subsequently reestablished upon tissue washout within 60 min. The interval between drug applications was at least 30 min.

dose–response curve for the amino acid–induced hyperpolarizing response is 8–10-fold left-shifted in the presence of a ﬁxed low concentration of baclofen (5 mM), suggestive of positive allosteric modulation. So far, L-Gln is the most potent natural amino acid found to potentiate baclofen responses in rat neocortical slices. Of interest, the concentration of L-Gln in the cerebrospinal ﬂuid is much higher than that of L-Asn or L-Orn. Yet, the effective concentration of L-Gln in potentiating GABAB receptor–mediated responses in rat brain slices (EC50 of 0.2 mM; n ¼ 12) is very much lower than that found in the cerebrospinal ﬂuid (97–99), whereas the potentiating concentrations of L-Orn (EC50 of 1 mM; n ¼ 12) and L-Asn (EC50 of 10 mM; n ¼ 12) are closer to those found in the cerebrospinal ﬂuid. This suggests that the L-Gln concentration in the synaptic cleft is, in fact, normally very low, presumably due to active uptake. HYPERPOLARIZING EFFECTS OF AMINO ACIDS IN RAT NEOCORTICAL SLICES L-Gln, L-Orn,

and L-Asn not only potentiated baclofen responses, but also induced rapid hyperpolarizing responses when applied alone, as shown for L-Glu (5 mM) in Figure 5. Both L-Orn and L-Asn also elicited membrane hyperpolarizations that lasted for about three to ﬁve minutes; the EC50 values for the hyperpolarization induced by each of these amino acids alone were estimated at 1.2 mM for L-Gln, 6 mM for L-Orn, and 10 mM for L-Asn. As far as we are aware, these amino acids have never been examined previously for activity at GABAB receptors. Certainly, if the responses arise from some other receptor, it must be one coupled to inwardly rectifying potassium channels, as they are sensitive to barium (100–300 mM; data not shown). We favor the notion that these responses are generated by the amino acids acting at GABAB2 subunits, though whether they act at the heptahelical domain, or at the VFT, remains unresolved.

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Figure 5 A representative trace showing the potentiating interactions between the GABAB receptor antagonist Sch 50911 (10 mM) and the hyperpolarizing response induced by L-Gln (5 mM), with a partial recovery of the control response to L-Gln upon drug washout. Abbreviation: GABAB, c-aminobutyric acidB.

INTERACTIONS BETWEEN AMINO ACIDS AND Sch 50911 In order to see if the hyperpolarizing responses induced by L-Gln or L-Orn were due to their activity at GABAB1 receptors, they were examined in the presence of the GABAB receptor antagonist Sch 50911 (100), which when applied on its own, did not affect the membrane potential. However, surprisingly, when Sch 50911 (10 mM) was coadministered with L-Gln (5 mM), there was a substantially potentiated response (Fig. 5). Notably, Sch 50911 not only increased the amplitude of the response to L-Gln by threefold, but also the onset of the response with L-Gln was markedly accelerated, followed by a rapid return of the response to the baseline upon washout. Similarly, L-Orn (5 mM), coadministered with Sch 50911 (10 mM), again gave a markedly increased amplitude of the L-Orn response, with accelerated onset and return to the baseline. With either L-Gln or L-Orn, control hyperpolarizing responses recovered within 30 minutes after washout of Sch 50911. The concentration–response curves for L-Gln and L-Orn were shifted threefold to the left by Sch 50911, indicative of an allosteric potentiation. Application of barium (200 mM) completely abolished the hyperpolarizing responses to L-Gln or L-Orn, as well as the responses in combination with Sch 50911, indicating that the responses are mediated through activation of inwardly rectifying potassium channels. However, GABAB receptor antagonists such as CGP 46381 (10–50 mM) had no such effect, showing that they did not originate from GABAB1 receptors. And yet, there was a

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threefold potentiating effect when Sch 50911 was coapplied with either L-Gln or L-Orn. Since there was no response to the antagonist Sch 50911 when used alone, there can be no associated downstream effect in its presence when coapplied with the amino acid. Thus, the large response to Sch 50911, when used in combination with the amino acids, can be attributed to some allosteric action of the amino acids on the GABAB receptor heterodimer, when the subunit GABAB1 is occupied by Sch 50911. This immediately recalls the conversion of Sch 50911 from an antagonist to a partial agonist in the heterodimer construct where the GABAB2 subunit also bears the VFT binding site from GABAB1 (71). Presumably, in our studies, the amino acids stimulate GABAB2 and allosterically modify the GABAB1 subunit, so that Sch 50911 acting at the latter subunit becomes converted to a full, potentiated agonist. Obviously, modulators acting at the GABAB2 subunit in turn allosterically modulate the GABAB1 subunit of the heterodimer, and vice versa. ALLOSTERIC INTERACTIONS AT FAMILY 3 GPCRs Our modulatory effects at GABAB receptors with amino acids and Sch 50911 recall a similar positive allosteric modulation of mGluR2 receptors by LY 487379 (44), where the partial agonists (2S,20 R,30 R)-2(20 ,30 -dicarboxycyclopropyl)glycine (DCG-IV) and (2S,10 S,20 S)-2-(carboxycyclopropyl)glycine (LCCG-1) both become potentiated to 150% of control glutamate maximal response, in the presence of LY 487379. In the latter case, it is suggested that the potentiated response is due to increased coupling to G-proteins, with the binding site for the modulator being at TM4 and TM5 of the mGluR2; indeed it is maintained (44) that an increase in agonist afﬁnity alone cannot explain the conversion of a partial agonist to a full agonist. There are other examples of such allosteric modulation; for instance, the antagonist 4-CHPG is converted to a partial agonist at mGluRs, by both DFB and N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide (CPPHA) (47). Neither of these positive modulators shows any effect on glutamate or quisqualate binding at these receptors, but simply bind at the heptahelical domain to modulate the receptor allosterically. It is of interest that the actions of allosteric modulators that increase agonist potency and efﬁcacy at the mGluR5 receptors, yet have no effect on agonist binding, can be appropriately modeled (47). For this, a modiﬁed model for allosteric modulation by Hall (45) was employed. It was concluded that the modulators can increase the activation cooperativity (d), resulting in an increased fraction of activated, agonist-bound mGluR5 receptor coupled to the G-protein, without affecting agonist binding. The dose–response curves generated for the modulators (47) bear a strong resemblance to those we found for the GABAB receptors, in the presence of the

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phenylalkylamines and the amino acids [Kerr et al. (56), (Figs. 3 and 4) and Kerr and Ong (79), (Fig. 2)]. This suggests that amino acids elicit a similar mechanism at the more complex heteromeric GABAB receptor. There is already considerable cooperativity in the coupling of GABAB2 receptors to the G-proteins, and in the presence of the potentiators, phenylalkylamines or amino acids, the Hill coefﬁcient becomes even higher, near two to three, with steeper dose–response curves. This is also true for the modulation of mGluR4 receptors by PHCCC, where the resemblance among the family of curves for potentiation at the mGluR4 receptor [Marino et al. (39), (Fig. 3)] is again very strong for our families of curves at GABAB receptors (56,79). ALLOSTERIC INTERACTIONS AT GABAB RECEPTORS Urwyler et al. (93) objected to our use of the term ‘‘allosteric’’ modulation in relation to the potentiating action of these amino acids or dipeptides, as well as phenylalkylamines, at GABAB receptors, averring that ‘‘this kind of potentiating effects (sic) in brain slice preparations might also well be due, for example, to downstream effects or receptor–receptor interactions, without the involvement of true allosteric mechanisms at the molecular level.’’ But for this to happen, the modulator must be activating some other receptor. Yet there are no responses to phenylalkylamines or many amino acids on their own, and therefore no downstream action. Also, amino acids and baclofen can activate inward-rectiﬁer potassium-dependent hyperpolarizations that are strongly superadditive when the two agents are combined. In other studies, when two independent agents, each individually inducing such inward-rectiﬁer responses, are combined, there is little or no superadditivity, but rather occlusion (23,101). For instance, adenosine and baclofen are also not fully additive in inducing an inward potassium current [see Figure 6 in Gassmann et al. (70)], suggesting that these agonists activate the same potassium channels. In our work, the superadditivity in the presence of combined agonist and modulators is thus evidence for modulation. Nevertheless, one possibility is an action of baclofen on some GABAB receptor function other than inward rectiﬁers, as with a GABAB receptor–mediated potentiation of mGluR (102), but this is unlikely in our preparations since inward-rectiﬁer actions predominate. One other effect that might contribute to our potentiated responses is the abolition of desensitization in GABAB receptors by cyclic adenosine monophosphate (cAMP) at the receptor (103). Such an effect could explain the prolongation of responses that we see, but not the marked enlargement. Therefore, we prefer a role for allosteric modulation in the potentiation effects observed. All the other examples of nonallosteric potentiation suggested in Urwyler et al. (93) refer to cAMP production by GABAB receptors (104), but not to inward-rectiﬁer potassium current activation; they are therefore also discounted in the present context. Rather, we favor some

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such explanation as that of Kenakin (105), where modulation need not be detectable in a particular assay, yet the receptor conformation can have changed. SUMMARY AND CONCLUSIONS Phenylalkylamines and amino acids positively modulate GABAB receptor– mediated actions in rat neocortical slice preparations. These modulators probably act at the heptahelical domain of the GABAB2 subunits where the modulator CGP 7930 acts as a partial agonist. It is known that stabilizing actions at both the extracellular and heptahelical domains will cause an increased afﬁnity and efﬁcacy of the heterodimer (106). Alternatively, cooperative interaction between GABAB receptors and calcium-sensing receptors could yield potentiation (107). Allosteric modulation seems to be a universal property among Family 3 receptors. These allosteric modulators uniformly bind at a groove on the extracellular face on the receptor heptahelical domains, as in mGluRs and calcium-sensing receptors. The amino acid sequences necessary for such binding at the heptahelical domain are also found in GABAB receptors, where arylalkylamines may bind to act as positive modulators. Amino acids often modulate Family 3 receptors where they bind at the VFT region, notably the calcium-sensing receptors. These binding sequences derive from the VFT of bacterial periplasmic amino acid binding proteins from which the receptors are ultimately derived. Indeed, the sequences for binding a-amino acids may be a characteristic feature in many GPCRs (108). Thus, a-amino acids may well bind at the VFT of the GABAB2 receptor; for example, L-Gln, L-Asn, and L-Orn themselves elicit powerful hyperpolarizing responses at GABAB receptors where they are also potentiators. It has been objected (93) that neither the phenylalkylamines nor the amino acids can be true allosteric modulators, since they do not inﬂuence ligand binding at the VFT of GABAB1 receptor (109). However, to the contrary, an increased receptor efﬁcacy can occur with modulators, yet without changing agonist-binding afﬁnity (47). Amino acids can potentiate the action of GABA or baclofen at recombinant GABAB1/2 heterodimers, which is best be interpreted as an allosteric action. Furthermore, L-Gln converts the GABAB receptor partial agonist/antagonist Sch 50911 to a potentiated agonist. A similar action with other partial agonist/antagonists is seen with CGP 7930 (Chapter 11). The most parsimonious explanation of all these results is that amino acids are indeed allosteric modulators at GABAB receptors. Both CGP 7930 and GS 39783 are potentiators at GABAB receptors in vivo (110,111). Unfortunately, little is known of their pharmacokinetics, and their therapeutic potential is so far uncertain, as is that of the compounds of the 3,Cl,4-OMe-fendiline type, which potently potentiate GABAB receptors (112). Certainly, these possibilities offer a stimulus to

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the development of clinically active allosteric positive modulators at GABAB receptors, in order to avoid overdosage or desensitization with baclofen. Positive modulators might also offer a favorable approach to boosting endogenous GABA-mediated actions at GABAB receptors in the clinic, particularly intrathecally for pain.

ACKNOWLEDGMENTS We are grateful to Professor Rolf H. Prager (Flinders University, South Australia) for his continuing collaboration in the synthesis and development of the compounds over the years. We also thank Dr. Mary Collins (University of Sydney) for her collaboration in the Xenopus oocytes experiments. Finally, we thank Dr. Wolfgang Froestl (Novartis, Switzerland) for the gifts of the various CGP compounds over the years, and Dr. David Blythin (Schering-Plough, U.S.A.) for the gift of Sch 50911.

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57. Kerr DIB, Ong J, Puspawati NM, Prager RH. Allosteric modulation: a new perspective in GABAB receptor pharmacology. Curr Top Pharmacol 2004; 8:17–29. 58. Conigrave AD, Quinn SJ, Brown EM. L-Amino acid sensing by the extracellular Ca2þ-sensing receptor. Proc Natl Acad Sci USA 2000; 97:4814–4819. 59. Zhang Z, Jiang Y, Quinn SJ, Krapcho K, Nemeth EF, Bai M. L-Phenylalanine and NPS R-467 synergistically potentiate the function of the extracellular calcium-sensing receptor through distinct sites. J Biol Chem 2002; 277: 33,736–33,741. 60. Miedlich S, Gama L, Breitwieser GE. Calcium sensing receptor activation by a calcimimetic suggests a link between cooperativity and intracellular calcium oscillations. J Biol Chem 2002; 277:49,691–49,699. 61. Young SH, Rozengurt E. Amino acids and Ca2þ stimulate different patterns of Ca2þ oscillations through the Ca2þ sensing receptor. Am J Physiol Cell Physiol 2002; 282:C1414–C1422. 62. Kerr DIB, Ong J. GABAB receptors. Pharmacol Exp Ther 1995; 67:187–246. 63. Ong J, Kerr DIB. Recent advances in GABAB receptors: from pharmacology to molecular biology. Acta Pharmacol Sin 2000; 21:111–123. 64. Kerr DIB, Ong J. Metabotropic GABAB receptors: new challenges in drug design. Curr Med Chem 2001; 1:27–42. 65. Bettler B, Kaupmann K, Mosbacher J, Gassmann, M. Molecular structure and physiological functions of GABAB receptors. Physiol Rev 2004; 84: 835–867. 66. Vacher C-M, Bettler B. GABAB receptors as potential therapeutic targets. Curr Drug Target CNS Neurol Disord 2003; 2:248–259. 67. Galvez T, Pin JP. How do G-protein-coupled receptors work? The case of metabotropic glutamate and GABA receptors. Med Sci 2003; 19:559–565. 68. Pin J-P, Kniazeff J, Goudet C, et al. The activation mechanism of class-C G-protein coupled receptors. Biol Cell 2004; 96:335–342. 69. Kaupmann K, Schuler V, Mosbacher J, et al. Human gamma-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying Kþ channels. Proc Natl Acad Sci USA 1998; 95:14,991–14,996. 70. Gassmann M, Shaban H, Vigot R, et al. Redistribution of GABAB(1) protein and atypical GABAB responses in GABAB(2)-deﬁcient mice. J Neurosci 2004; 24:6086–6097. 71. Margeta-Mitrovic M, Jan YN, Jan LY. Ligand-induced signal transduction within heterodimeric GABAB receptor. Proc Natl Acad Sci USA 2001; 98: 14,643–14,648. 72. Liu J, Maurel D, Etzol, S, et al. Molecular determinants involved in the allosteric control of agonist afﬁnity in the GABAB receptor by the GABAB2 subunit. J Biol Chem 2004; 279:15,824–15,830. 73. Quiocho F, Ledvina PS. Atomic structure and speciﬁcity of bacterial periplasmic receptors for active transport and chemotaxis: variation of common themes. Mol Microbiol 1996; 20:17–25. 74. Sack JS, Saper MA, Quiocho FA. Periplasmic binding protein structure and function. Reﬁned X-ray structures of the leucine/isoleucine/valine-binding protein and its complex with leucine. J Mol Biol 1989; 206:171–191.

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91. Parker DAS, Ong J, Marino V, Kerr DIB. Gabapentin activates presynaptic GABAB hetero-receptors in rat cortical slices. Eur J Pharmacol 2004; 495: 137–143. 92. Urwyler S, Pozza MF, Lingenhoehl K, et al. NN0 -Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds: novel allosteric enhancers of c-aminobutyric acidB receptor function. J Pharmacol Exp Ther 2003; 307:322–330. 93. Urwyler S, Gjoni T, Kaupmann K, Pozza MF, Mosbacher J. Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABAB receptors. Eur J Pharmacol 2004; 483:147–153. 94. Ong J, Parker DAS, Marino V, Kerr DIB, Puspawati NM, Prager RH. 3-Chloro,4-methoxyfendline is a potent GABAB receptor potentiator in rat neocortical slices. Eur J Pharmacol 2005; 507:35–42. 95. Ferry S, Traiffort E, Stinnakre J, Ruat M. Developmental and adult expression of rat calcium-sensing receptor transcripts in neurons and oligodendrocytes. Eur J Neurosci 2000; 12:872–884. 96. Ong J, Bexis S, Marino V, Parker DAS, Kerr DIB, Froestl W. Comparative activities of the enantiomeric GABAB receptor agonists CGP 44532 and 44533 in central and peripheral tissues. Eur J Pharmacol 2001; 412:27–37. 97. Kato M, Jin H, Sakai-Kato K, Toyo’oka T, Dulay MT, Zare RN. Determination of glutamine and serine in rat cerebrospinal ﬂuid using capillary electrochromatography with a modiﬁed photopolymerized sol–gel monolithic column. J Chromatogr A 2003; 1004:209–215. 98. Rainesalo S, Keranen T, Palmio J, Peltola J, Oja SS, Saransaari P. Plasma and cerebrospinal ﬂuid amino acids in epileptic patients. Neurochem Res 2004; 29:319–324. 99. Nishimura F, Nishihara M, Mori M, Torii K, Takahashi M. Excitability of neurons in the ventromedial nucleus in rat hypothalamic slices: modulation by amino acids at cerebrospinal ﬂuid levels. Brain Res 1995; 691:217–222. 100. Ong J, Marino V, Parker DAS, Kerr DIB, Blythin DJ. The morpholino-acetic acid analogue Sch 50911 is a selective GABAB receptor antagonist in rat neocortical slices. Eur J Pharmacol 1998; 362:35–41. 101. Sodickson DL, Bean BP. Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors. J Neurosci 1998; 18:8153–8162. 102. Hirono M, Yoshioka T, Konishi S. GABAB receptor activation enhances mGluR-mediated responses at cerebellar excitatory synapses. Nature Neurosci 2001; 4:1207–1216. 103. Couve A, Thomas P, Calver AR, et al. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABAB receptor-effector coupling. Nat Neurosci 2002; 5:415–424. 104. Cunningham MD, Enna SJ. Evidence for pharmacologically distinct GABAB receptors associated with cAMP production in rat brain. Brain Res 1996; 720: 220–224. 105. Kenakin TP. The secret lives of GPCRs. Drug Discov Today 2003; 8:674. 106. Pin J-P, Parmentier M-L, Prezeau L. Positive allosteric modulators for c-aminobutyric acidB receptors open new routes for the development of drugs

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12 Muscarinic Receptors Targeting Allosteric Modulation by the Development and Application of the Radiolabeled Allosteric Agent [3H]dimethyl-W84 Christian Tra¨nkle Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Bonn, Bonn, Germany

INTRODUCTION During the last decade it became evident that in an increasing number of G-protein–coupled receptors neurotransmitter binding and action may be modulated by allosteric modulators interacting with an allosteric site on the extracellular face of the receptor topologically distinct from the orthosteric site (1). During the last 40 years mAChRs have been a paradigm for the simultaneous binding of two structurally different agents at distinct sites in a G-protein–coupled receptor leading to a ternary complex. In the ternary complex these agents may affect each other’s binding afﬁnity. These allosteric cooperative interactions offer a novel approach to get an insight into the molecular principles of ligand–receptor interactions and open the perspective for new strategies of drug therapy [for review see Refs. (2–7)]. Many features of allosteric modulation, e.g., deﬁned maximum effect on function, mapping of modulator receptor interactions, subtype selective modulation of orthosteric ligand binding at the neurotransmitter site, and 287

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data evaluation by means of the ternary complex model of allosteric interaction, were initially studied in muscarinic receptors using orthosteric radioligands. Archetypal M2 prevalent muscarinic allosteric agents known for many years include gallamine, alcuronium, and bis(ammonio) alkanetype agents such as W84. Until the development of the radioligand [3H]dimethyl-W84 (Fig. 1) for the allosteric site in muscarinic M2 receptors described in this review, the binding characteristics of allosteric agents had to be deduced from the effects they exerted on the binding of radioligands to the orthosteric site [e.g., Lazareno and Birdsall (8)]. Because of the lack of a radiolabeled allosteric agent it had not been possible to directly study the binding of agents at the allosteric site (3). Additionally, it had not been possible to reject by direct measurements the objection that allosteric actions on ligand-occupied muscarinic receptors might only be an aspect of a rather nonspeciﬁc attachment to cellular membranes, although abundant evidence had pointed to a speciﬁc and preferential interaction. With the development of [3H]dimethyl-W84 as an allosteric radioligand it was possible for the ﬁrst time to test the allosteric principle in a G-protein– coupled receptor in a rigorous way by directly addressing the ‘‘other side of the coin.’’ This review focuses on the development of the radioalloster [3H]dimethyl-W84 and highlights the different stages: addressing receptor speciﬁcity of allosteric action, choice of the modulator, target characterization, labeling the receptor by the radioalloster, testing the cooperativity model, and radioalloster application to elucidate different modes of allosteric actions. Finally, a mutagenesis approach, which led to a model of the M2 receptor generated by molecular modeling, is presented that allows visualizing of the allosteric binding site used by a number of allosteric modulators including dimethyl-W84. PRINCIPLES OF MUSCARINIC ALLOSTERIC ACTION In principle, the equilibrium binding of a given orthosteric ligand can be reduced, enhanced, or left unaffected depending on the type of allosteric agent, reﬂecting negative, positive, or neutral cooperativity between the allosteric agent and the orthosteric ligand. The equilibrium binding of an orthosteric ligand depends on the probability of ligand association and dissociation (9), which can both be affected by allosteric agents. This is shown in Figure 2 for the bis(ammonio) alkane-type allosteric agent dimethyl-W84. The binding of the radiolabeled muscarinic antagonist N-methylscopolamine ([3H]NMS) was measured in homogenates of domestic pig hearts (10). Dimethyl-W84 reduced both the association and the dissociation rate constant of the radiolabeled orthoster. A changed dissociation time course of a bound orthosteric ligand undoubtedly points to binding of an agent to an allosteric receptor site different from the orthosteric binding site and indicates ternary complex formation (8,11,12). Commonly,

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Figure 1 Structural formulas of selected allosteric modulators of muscarinic receptors. Dimethyl-W84 (encircled groups have been replaced by [3H]methyl-residues in the radioalloster), benzylidene-W84, W84, Chin3/6, and hexamethonium are bis(ammonio) alkane-type compounds; WDuo3, Duo3, and obidoxime are bispyridinium-type modulators. Gallamine is a terquaternary modulator; alcuronium and diallylcaracurine are bisquaternary compounds, whereas strychnine and tacrine (THA) are monoquaternary allosteric agents at physiological pH.

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Figure 2 Time course of (A) [3H]NMS association to and (B) [3H]NMS dissociation from M2 receptors in porcine cardiac membranes under control conditions and in the presence of the indicated concentrations of dimethyl-W84. Monoexponential curve ﬁtting. Ordinates (A, B): Speciﬁc binding of [3H]NMS. Abscissa: Time after the start of the respective kinetic process. The association was started by addition of [3H]NMS to M2 receptor–containing membranes that had been equilibrated with dimethylW84 for 30 minutes. The dissociation was visualized 30 minutes after mixing of the receptors with dimethyl-W84 by addition of 1 mM atropine. (C) Effects of dimethyl-W84 on [3H]NMS equilibrium binding and [3H]NMS kinetic constants of association (kþ1) and dissociation (k1). Left ordinate: Speciﬁc binding of 0.2 nM [3H]NMS as a percentage of control in the absence of dimethyl-W84. Mean values SEM from four experiments, performed in triplicate, are given. Curve ﬁtting to equilibrium data was performed applying the allosteric ternary complex model. Right ordinate: apparent rate constants of association (kþ1) and dissociaton (k1) as a percentage of the respective controls in the absence of dimethyl-W84. Means SEM of one to four independent experiments are given. Abbreviation: [3H]NMS, radiolabeled N-methylscopolamine. Source: From Ref. 10.

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muscarinic allosteric agents retard the dissociation of orthosteric ligands; rarely, they may accelerate it (13,14). Considered separately, an inhibition of ligand dissociation will enhance ligand equilibrium binding, whereas an inhibition of association will decrease equilibrium binding of the orthosteric ligand. There is only one report of an allosteric increase of ligand association at muscarinic receptors (15). Normally, ligand association is inhibited by allosteric agents; phenomenologically, they may therefore resemble competitive antagonists. The reduction of orthosteric ligand association is caused by the interaction of the allosteric agent with unoccupied receptors (Fig. 2); if it exceeds the inhibition of orthosteric ligand dissociation, the agent reduces orthosteric ligand equilibrium binding, as in the case of the allosteric agent dimethyl-W84 (Fig. 2). In other words, dimethyl-W84 has a higher afﬁnity for free than for NMS-occupied M2 receptors. The interaction between allosteric and orthosteric agents in muscarinic receptors has often been described by the ternary complex model of allosteric interactions (8,11,12) illustrated in Figure 3 (16), also frequently referred to as the ‘‘cooperativity model.’’ According to Ehlert (12), the measure of allosterism, i.e., the cooperativity factor a, corresponds to the ratio of the alloster binding constants for the interaction with orthoster-occupied receptors and the binding constant for free receptors; its value indicates whether the allosteric agent elevates, reduces, or leaves unchanged the equilibrium binding of the orthosteric ligand corresponding to positive, negative, and neutral cooperativity, respectively. One reason for the development of the radiolabeled allosteric ligand [3H]dimethyl-W84 was the prospect of testing the cooperativity model by direct binding measurements at the common allosteric site.

Figure 3 Ternary complex model of allosteric interactions according to Ehlert (12). X: orthosteric agent, R: receptor, A: allosteric modulator. Kx: equilibrium dissociation constant of the orthosteric ligand binding to free receptors, KA: equilibrium dissociation constant of the allosteric ligand binding to free receptors, a: cooperativity factor representing the reciprocal shift of the binding constants in the ternary complex XRA. The sketches visualize the corresponding states of site occupancy within the receptor protein. Source: From Ref. 16.

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MUSCARINIC RECEPTOR SPECIFICITY OF ALLOSTERIC ACTIONS The starting point for the development of a radioactive modulator was the studies in which it was investigated whether (i) the muscarinic allosteric actions were receptor mediated, (ii) independent of G-protein coupling, and (iii) speciﬁc for muscarinic compared with other G-protein–coupled receptors. Phospholipid Interaction Hypothesis Many agents possess the ability to perturb receptor conformations by nonspeciﬁcally interacting with the surrounding lipid bilayer. Since it is known that cationic amphiphilic agents like most allosteric modulators tend to interact with membrane interphases [e.g., Girke et al. (17)], it could be objected that allosteric actions originate from a nonspeciﬁc attachment of these modulators to membrane surfaces. Using phospholipid monolayers as an interphase model, the displacement of 45Ca2þ [e.g., Klein et al. (18)] to test for a membrane interaction of a large series of allosteric modulators was measured. The potency of the modulators to displace 45Ca2þ binding and the potency to retard allosterically the dissociation of [3H]NMS from M2 receptors did not correlate (16,19). However, a number of agents such as quinidine, verapamil, and D-tubocurarine showed equal potencies in both assays and thus indicated that their allosteric action is not a preferential event. The archetypal allosteric agents W84 (20), gallamine (21), and alcuronium (22) were among the agents whose allosteric action far exceeded their perturbing effect on the interphase (16). Dimethyl-W84 showed the least tendency to interact with the membrane interphase (16,19). Therefore, its allosteric effect is not likely to be due to a nonspeciﬁc adsorption phenomenon. Specificity of G-Protein Coupling of Muscarinic M2 and M3 Receptors and Their Sensitivity Toward Allosteric Modulation It is known that certain polyanionic compounds interfere with G-protein coupling via interaction with the receptor–G-protein interface (23) and may enhance antagonist binding by a yet unknown mechanism (24). Among the ﬁve muscarinic receptor subtypes M1 to M5, the allosteric sensitivity of the M2 and M4 subtypes is very pronounced (14,25–27). Both M2 and M4 receptors couple preferably to inhibitory G-proteins, whereas M1, M3, and M5 receptors preferably interact with stimulatory G-proteins (28,29). In the past it had repeatedly been questioned whether the high allosteric sensitivity of M2 receptors toward allosteric modulation is related to its coupling selectivity (14,27). The third intracellular loop i3 is part of the receptor–G-protein interface and determines to a great extent the coupling speciﬁcity of a receptor–G-protein interaction (30–32). The i3 loop of the

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M2 receptor represents a considerable part of the receptor protein (33). If the i3 loop is important for the high allosteric sensitivity of the M2 subtype and the low sensitivity of the M3 subtype, an exchange of the i3 loop between the two subtypes should decrease the allosteric sensitivity of M2 and, conversely increase that of the M3 subtype. The allosteric actions of the archetypal modulator gallamine on the dissociation and equilibrium binding of the orthosteric radioligand [3H]NMS in wild-type M2, M3 and chimeric M2–M3 receptors with exchanged i3 loops were unaffected by the mutation (34). These data spoke against a relationship between receptor– G-protein–coupling selectivity and the divergent sensitivity of M2 and M3 receptors towards allosteric modulation by gallamine. The ﬁndings suggested that the different sensitivities of muscarinic receptors to allosteric modulation are based on subtype-speciﬁc differences in the amino acid composition of the allosteric sites. Specificity of Muscarinic Allosteric Modulators for Muscarinic Receptors Allosteric effects of W84, gallamine, and alcuronium on the dissociaton of an orthosteric ligand were absent in H1-histamine (35), ß-adrenergic, and A1-adenosine receptors (36). Thus, their allosteric action is not a common feature among G-protein–coupled receptors. However, alcuronium and gallamine, at higher concentrations, accelerated the dissociation of the a1-adrenergic antagonist [3H]prazosine and partially inhibited its equilibrium binding, suggesting an allosteric action (36). [3H]prazosine binding has also been reported to be inhibited by brucine, a muscarinic allosteric enhancer of McN-A-343 action at M1 receptors (37); brucine resembles about one-half of the alcuronium molecule. In conclusion, the muscarinic speciﬁcity of the alkane-bis-ammonium–type modulator W84 exceeds that of other archetypal muscarinic allosteric agents.

SEARCH FOR A POTENTIAL ALLOSTERIC RADIOLIGAND OF M2 RECEPTORS At the beginning of the search for a potential allosteric radioligand, there was the question of whether there exist compounds whose main pharmacological action was allosteric and which by a nanomolar afﬁnity fulﬁll the prerequisite to be employed as a radioligand in direct binding measurements. A variety of compounds from different pharmacological groups had been studied for their allosteric effects, such as alcuronium [e.g., Refs. (38–41)], methoctramine (42,43), D-tubocurarine (38,44–46), tacrine (47,48), quinidine (49), verapamil [e.g., Refs. (50,51)], and obidoxime (52,53). Most of these agents have another predominant pharmacological

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action. The existing data from the literature could not be directly compared because (i) they differed with respect to the protocol used to detect allosteric actions [e.g., residual binding curves (27)], or inhibition of the rate constant of radioligand dissociation (14)]; (ii) the allosteric potency depends on the assay conditions [e.g., Pedder et al. (54)], which were divergent among the various groups of investigators; and (iii) the measures used to deﬁne allosteric potency differed among the groups (9), e.g., an EC50,diss value [indicating a reduction of the dissociation rate constant k1 to 50%, equivalent to p(a KA) in the ternary complex model, cf. Fig. 3), was not always reported. Therefore, at the beginning of the search for a potential radioligand an inventory was established to deﬁne a rank order of the allosteric potency of a number of structurally different compounds from various pharmacological groups under conditions that ensured a high allosteric potency (55). This comparison should help to identify lead structures with a high binding afﬁnity. For the development of a radiolabeled modulator it was essential to ﬁnd an agent whose main action was allosteric and that by chemical modiﬁcation could be derivatized to yield an agent with a nanomolar allosteric potency necessary for its employment in direct binding measurements (56). Because allosteric actions and quantitative structure activity relationships (QSARs) depend on the buffer system used (55), in most of the following studies a Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 C) of low ionic strength without divalent cations was applied, which is known to provide high sensitivity of M receptors toward allosteric modulation (14). A ranking of allosteric modulator is given in Table 1. First, the effect of the top-ranked modulators dimethyl-W84 and ‘‘diallylcaracurine-V,’’ a derivative of alcuronium, occurs in the nanomolar concentration range, pointing to a highly complementary binding of these agents to the allosteric site of muscarinic M2 receptors. Second, the allosteric potency was rather sensitive to structural modiﬁcations. In the alkane-bis-ammonium compound W84 (EC50,diss Na,K,Pi: 24 nM), the phthalimide rings seem to be essential for a high allosteric potency since hexamethonium, which in W84 is contained as a spacer, has a low potency (EC50,diss Na,K,Pi: 114.000 nM). In a similar way, the spacer of the bispyridinium-type modulators, e.g., WDuo3 and Duo3, seems to be uncritical, because the closely related bispyridinium-type modulator obidoxime is only weakly potent. At this stage of radioalloster development in the literature, parameters characterizing the allosteric potency of highly potent allosteric agents were few and none of these agents had a nanomolar afﬁnity: Ellis et al. (14,43,53) reported on the potency of gallamine, D-tubocurarine, tacrine, DL-verapamil, and obidoxime to retard allosterically the dissociation of [3H]NMS from M2 receptors in 5 mM Na,K,Pi buffer; these values were in line with the data given in Table 1. Lee and El-Fakahany (27) compared the allosteric action of chinidine, verapamil, gallamine, and methoctramine in 50 mM Na,K,Pi buffer. In this study methoctramine was the most potent compound with respect to

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Table 1 Choice of a Potential Radioligand from a Ranking of Allosteric Modulators of Muscarinic M2 Receptors Na,K,Pi

Mg,Tris,Cl,Pi

EC50,diss (nM)

nH

EC50,diss (nM)

Dimethyl-W84 (10), diallylcaracurine-V Alcuronium Benzylidene-W84 (57) Tert. W84 (58) Tetra-W84 C730 -phth WDuo3 Chin3/6 W84 Methoctramine Duo3 Tacrine (THA) D-Tubocurarine Gallamineb

3

1.0

170

0.9

57

4 16 n.d. 7 11 19 62 24 760 1,300 3,100 860 180 EC53 ¼ 160 n.d. 4,000 8,500 76,000 EC59 ¼ 42,000 114,000 EC61 ¼ 66,000 91,000

1.2 1.2 n.d. 2.1 1.0 1.1 1.4 1.1 1.5 2.6 3.0 1.1 1.1

55 70 240 310 (9) 390 480 (59) 750a (60) 1,300 (59) 3,000 4,200 (61) 48,000 11,000 16,000

1.2 1.2 1.0 1.1 1.0 1.4 1.4 1.0 1.0 1.7 1.8 1.1 1.0

14 4 n.d. 15 36 25 12 54 4.0 3.2 16 13 89

n.d. 0.8 1.0 0.7

22,000a (62) 22,000 31,000 >100,000 (52)

0.9 1.1 0.9 n.d.

n.d. 5.5 3.6 n.d.

1.0

n.d.

n.d.

n.d.

0.9

n.d.

n.d.

n.d.

AF-DX 384 Chinidine DL-Verapamil Obidoximeb Hexamethoniumb Atropine

nH

Potency ratio

Compound

Note: Allosteric potency of the test compounds to retard the dissociation of radiolabeled N-methylscopolamine ([3H]NMS) from porcine cardiac M2 receptors at the indicated incubation conditions. a Guinea pig myocardium. b Curves leveled off above zero; the concentration at the inﬂection point is additionally indicated, the corresponding level of k1 is indicated by the respective subscript of EC. Deﬁnitions of terms: Na,K,Pi: 4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 C; Mg,Tris,Cl,Pi: 3 mM MgHPO4, 50 mM Tris–HCl, pH 7.3, 37 C; EC50,diss: concentration (in nM), reducing the apparent rate constant of [3H]NMS dissociation k1 to 50% of the control value; nH: slope factor of the concentration effect curves; potency ratio: EC50diss,MgTrisClPi/EC50diss,NaKPi; n.d.: not determined. Source: From Ref. 55.

the retardation of [3H]NMS dissociation; however, EC50,diss values were not determined. Attractive lead structures for the development of new allosteric agents emerged such as phthalimide-substituted bisquaternary ammonium

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compounds and structures related to alcuronium. Introduction of a methyl group in the phthalimide rings of W84 increased the allosteric potency by a factor of eight in the low nanomolar concentration range, holding out the prospect of direct binding measurements with the tritiated compound (Table 1). More recently, other bis(ammonio) alkane derivatives have been found with an even higher afﬁnity at NMS-occupied (and free) M2 receptors (63), some of them showing positive cooperativity with NMS (16,64). Alcuronium, which is ranked equally with dimethyl-W84 in Table 1, is an enhancer of the binding of some muscarinic agonists (65) and antagonists (38,39). This stimulated others to synthesize derivatives of brucine, which can be regarded as an alcuronium molecule split in part. Some of them show positive cooperativity with acetylcholine (66); however, generally, the halving of the molecule is accompanied by a signiﬁcant loss of allosteric potency (8,66). It may be considered as a disadvantage that brucine interacts with glycine receptors and aluronium has neuromuscular blocking activities at allosterically relevant concentrations. 4,40 -Diallylcaracurine-V (‘‘caracurine-V’’) is an alcuronium derivative that is hydrolytically more stable than the mother compound (67) and has a substantially decreased neuromuscular blocking activity and toxicity in mice (up to 20 mg/kg body weight inactive) (68) compared to the mother compound lethal dose, 50% (LD50) ¼ 0.2 mM/kg, equivalent to 0.15 mg/kg (69). Diallylcaracurine-V has the same allosteric effects on the kinetics and equilibrium binding of [3H]NMS as alcuronium (70). The stereochemical structure of the rigid diallylcaracurine-V ring system has been solved (71); caracurine-V derivatives show allosteric effects similar to alcuronium with respect to afﬁnity and positive cooperativity with NMS (72) as do iso caracurine-V and tetrahydrocaracurine-V derivatives (73). We chose dimethyl-W84 as a candidate for the development of a radioalloster because, ﬁrst, the EC50,diss value of 3 nM meant a top position for dimethyl-W84 in the modulator ranking; second, dimethyl-W84 was easily accessible by chemical synthesis; and third, the toxicity of the mother compound W84 in mice was known to be low (LD50 ¼ 44 mmol/kg body weight, equivalent to 0.31 mg/kg) (74), with a mortality rate of 5% to 10% among mice who received a dose of 3 mmol/kg body weight. Thus, the mother compound of dimethyl-84 is tolerated in vivo at doses that lead to allosterically relevant plasma concentrations. BINDING TOPOLOGY OF ALLOSTERIC MODULATORS IN MUSCARINIC M2 RECEPTORS Another desirable, but not compelling, prerequisite for the development of an allosteric radioligand was that there should be for a number of structurally different allosteric agents a commonly used allosteric site. The labeling of such a site by a radioalloster would then allow a representative investigation

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of this principle of pharmacological action. Since the beginning of the 1990s there has been evidence for a common allosteric site on muscarinic M2 receptors: Ellis and Seidenberg (53) demonstrated that the allosteric retardation of the dissociation of [3H]NMS from M2 receptors caused by gallamine is antagonized by obidoxime in a competitive fashion. The allosteric actions of 8-(NN-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB8) and tacrine were also inhibited by obidoxime but the type of antagonism had not been analyzed (53). With regard to the equilibrium binding of [3H]NMS, Waelbroeck (45) observed a competitive interplay between the effects of the modulators d-tubocurarine and gallamine. Similarly, the effects of combinations of alcuronium and gallamine, which elevate and reduce [3H]NMS equilibrium binding, respectively, could be predicted by Prosˇka and Tucˇek (75) on the basis of a model for a competitive interaction. Applying the experimental approach introduced by Ellis and Seidenberg (53) and using obidoxime as an antagonist it was found that the allosteric actions of dimethyl-W84 as well as a number of structurally divergent cationic modulators including gallamine, alcuronium, W84, WDuo3, and a benzylidene-substituted W84 derivative were competitively antagonized with similar potency, supporting their interaction with the common allosteric site (Fig. 4) (81). This site was believed to be located at the entrance of the ligand binding pocket of the receptor protein (62,82). The fact that the benzylidenesubstituted W84 derivative showed a ‘‘common site behavior’’ (83) provided evidence for a considerable spatial extension of the allosteric site. If there were only one allosteric binding site, obidoxime would interfere with the allosteric actions of all modulators in the same way. On the other hand Duo3 (Fig. 1) stood out in being much less sensitive to obidoxime and in being antagonized by obidoxime in a manner not compatible with a competitive interplay, leading to the hypothesis of a second allosteric site (Fig. 4B) (81). Similarly, structure–activity relationships in bispyridinium-type model compounds including Duo3, which are potent allosteric modulators of muscarinic M2 receptors (59,61), were not readily compatible with the common site model and were interpreted to point to a multiple binding mode of bisquaternary allosteric modulators (59). In the meantime, a second allosteric site on muscarinic receptors, which exhibits a pharmacology different from ‘‘typical’’ allosteric modulators, has also been described for derivatives of staurosporine (84,85). Taken together, the sensitivity of dimethyl-W84 to obidoxime and the competitive character of this interaction suggested the tritiated modulator to be suitable for a direct labeling of the common allosteric site of the M2 receptor. MUSCARINIC SUBTYPE SELECTIVITY OF DIMETHYL-W84 Whereas the binding site for the orthosteric ligand acetylcholine is highly conserved among the ﬁve muscarinic receptor subtypes (86), the allosteric

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Figure 4 (A) Effects of dimethyl-W84 and obidoxime, alone or in combination, on the time course of [3H]NMS dissociation. Ordinate: Speciﬁc [3H]NMS binding as a percentage of the binding before the start of the dissociation. Abscissa: Time after initiation of [3H]NMS dissociation by addition of atropine alone or in combination with the indicated compounds. Obidoxime reduces the stabilizing action of dimethyl-W84 on [3H]NMS–M2 receptor complexes. (B) Results of the nonlinear analysis according to Lew and Angus (open symbols) or Waud (closed symbols), which were transformed to dose ratios (DR) ¼ (EC0.5,obidoxime/EC0.5,control) and are linearly illustrated according to Arunlakshana and Schild. Abbreviation: [3H]NMS, radiolabeled N-methylscopolamine. Source: From Ref. 77–81.

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site is likely to be less well conserved than the orthosteric ligand binding site, thus potentially allowing the design of ligands with greater subtype selectivity. Remarkably, muscarinic allosteric ligands generally exhibit the highest afﬁnity to the M2 receptor subtype (14,87–89). An interaction with both the orthosteric and the allosteric site in M2 receptors has been postulated for the M2 selective muscarinic antagonists methoctramine (90) and AF-DX 384 (62): the interaction of these antagonists with the orthosteric site is believed to govern their afﬁnity, whereas the interaction with the allosteric site is believed to govern the M2 selectivity of these compounds. An advantage of allosteric modulators is that their subtype selectivity is based not only on their receptor afﬁnity but also on their cooperativity with the orthosteric ligand. Thus, allosteric modulation of muscarinic receptors offers the possibility of increasing the action of orthosteric agonists with an extent of subtype selectivity not achieved before (91,92). Even absolute subtype selectivity becomes possible if an agent showing positive or negative cooperativity with, e.g., the endogenous ligand at one receptor subtype and neutral cooperativity at the other subtypes would exert functional effects at only the one subtype, regardless of the concentration of agent or its afﬁnities for the subtypes (93). During the development of dimethyl-W84 as a potential radioligand, the afﬁnity of the modulator with free and NMS-occupied M1–M5 receptors, as well as the cooperativity between dimethyl-W84 and NMS, were determined at all subtypes (94). Dimethyl-W84 fully inhibited the dissociation rate of [3H]NMS at all subtypes. The pEC50,diss value was taken as a measure of the binding constant at NMS-occupied receptors (Table 2). [3H]NMS equilibrium binding at M1–M5 receptors was partially inhibited by dimethyl-W84. Data analysis using the allosteric ternary complex model gave the log cooperativity factor pa characterizing the interaction between dimethyl-W84 and

Table 2 Subtype Selectivity of Dimethyl-W84 M1

M2

M3

M4

M5

pKA 7.08 0.11 8.65 0.04 6.14 0.06 6.92 0.01 5.57 0.28 pa 0.13 0.03 0.58 0.01 0.43 0.15 0.10 0.01 0.52 0.31 pEC50,diss 6.89 0.04 7.96 0.07 5.81 0.05 6.82 0.02 5.16 0.05 Deﬁnitions of terms: pKA: negative log value of the equilibrium dissociation constant for the binding of dimethyl-W84 to the respective muscarinic receptor subtype derived from the cooperative interaction with the orthosteric ligand [3H]NMS (radiolabeled N-methylscopolamine) according to Ehlert (12). pa: negative log cooperativity factor for the allosteric interaction between A and L (with a < 1, a > 1, a ¼ 1 indicating positive, negative, and neutral cooperativity, respectively). pEC50,diss: negative log concentration of the modulator that reduces the rate constant of [3H]NMS dissociation by 50%; according to the ternary complex model pEC50,diss equals p(KA a).

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Figure 5 Binding constants of dimethyl-W84 in free M1–M5 muscarinic receptor subtypes compared to other subtype selective compounds. Source: From Ref. 76.

NMS and the negative log equilibrium dissociation constant (pKD) for dimethyl-W84 binding to free receptors. The rank order of afﬁnities for free receptors and NMS-occupied receptors (M2 > M1 ¼ M4 > M3 > M5) was the same. At all subtypes, dimethyl-W84 exhibited weak negative cooperativity with NMS; thus, the afﬁnity to free receptors was always smaller than to NMS-occupied receptors. Remarkably, dimethyl-W84 is an M2-preferring muscarinic allosteric ligand with a 600- to 1000-fold M2 to M5 selectivity that is greater than that of other M2 selective antagonists (Fig. 5). Having in mind that there is experimental evidence from structurally diverse allosteric agents including W84 that these agents in free muscarinic M2 receptors still bind solely to the allosteric site (45,95–97) and do not utilize the orthosteric site, exclusive interaction of dimethyl-W84 with the allosteric site obviously confers a high binding afﬁnity.

FUNCTIONAL EFFECTS OF DIMETHYL-W84 IN M2 RECEPTORS Dimethyl-W84 behaves as an allosteric antagonist with a pronounced negative cooperative effect with the agonist oxotremorine (98). In contracting guinea pig left atria the concentration–effect curve for the negative inotropic action of oxotremorine M was shifted to the right by dimethyl-W84 in a parallel fashion. The Schild plot revealed a slope of unity up to the highest

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applied dimethyl-W84 concentration of 105M. The pKA as a measure of alloster afﬁnity to the free receptor amounted to 7.70, which is equivalent to KA ¼ 2 108 M. Gallamine is known to exert allosteric effects also in isolated guinea pig atria (21). In the same model, alcuronium antagonized the action of the oxotremorine M in a formally competitive way (99). It should be noted that for W84, WDuo3, and Chin3/6, the allosteric retardation of [3H]NMS dissociation has also been demonstrated under organ bath conditions in contracting guinea pig left atria (52,100). Taken together, the potential radioligand dimethyl-W84, like other common site allosteric ligands, behaved as an allosteric muscarinic antagonist and exerted a high allosteric potency at M2 receptors (pKB ¼ 7.7) (98), comparable to that of the M2 selective antagonists methoctramine and AF-DX 384 (62). [3H]DIMETHYL-W84 AS A RADIOLIGAND FOR THE COMMON ALLOSTERIC BINDING SITE OF MUSCARINIC M2 RECEPTORS The synthesis of the corresponding radiolabeled compound [3H]dimethylW84 was carried out by Amersham Life Science (Braunschweig, Germany) starting off from the bistertiary compound. Methylation of both tertiary nitrogens was achieved by treatment of the tertiary dimethyl-W84 with an excess of tritiated methyliodide. The speciﬁc activity amounted to 168 Ci/ mmol ¼ 6.22 TBq/mmol, radiochemical purity was 97%. The stock solution contains ethanol–water–methanol 84:15:1. In mixtures of ethanol and water as well as in distilled water no chemical decomposition of the unlabeled compound could be detected. In the buffer of the initial studies (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 C) dimethyl-W84 undergoes slow hydrolysis with a half-life of t1/2 ¼ 16 hours, which is several fold beyond the applied incubation time. After the radiolabeling with tritium [3H]dimethyl-W84, the question of whether it is possible to measure a speciﬁc binding in native membrane preparations and whether the allosteric actions of dimethyl-W84 are mediated via the M2 receptor protein was addressed. The binding of [3H]dimethyl-W84 was studied in the same buffer of low ionic strength devoid of divalent cations in which the modulators had been ranked earlier (cf. Chapter 4) in order to provide optimum conditions for a high afﬁnity at the allosteric site in NMS-occupied M2 receptors (10). Since the kinetics of [3H]dimethyl-W84 binding at NMS-occupied receptors were expected to be very fast (81), a centrifugation procedure was applied to measure [3H]dimethyl-W84 binding in NMS-occupied and in free M2 receptors (10) under equilibrium conditions (101). In the initial studies a saturating concentration of NMS (1 mM) was applied to occupy the orthosteric site of the M2 receptors, because the ability to bind with rather high afﬁnity to ligand-occupied receptors undoubtedly

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distinguishes allosteric modulators from conventional ligands. Furthermore, experiments with [3H]NMS as the radioligand suggested that dimethyl-W84 binds with a slightly higher afﬁnity to free receptors than in the [3H]NMS-liganded receptor (cf. Fig. 2C). Since it cannot be ruled out that the orientation of dimethyl-W84 in the free receptor might be somewhat different than in NMS-occupied receptors, the orthosteric site was blocked by NMS. Finally, measurement of [3H]dimethyl-W84 binding

Figure 6 (Caption on facing page)

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in NMS-occupied receptors made possible a direct comparison with the functional dissociation delay experiments in which the allosteric actions of nonlabeled dimethyl-W84 and other modulators were characterized at [3H]NMS-liganded receptors. This allowed checking whether the site of dimethyl-W84 binding is identical with the site that mediates the actions of dimethyl-W84 and of the other applied allosteric agents. The highafﬁnity component of saturable [3H]dimethyl-W84 binding (Fig. 6A) probably represents occupation of the M2 receptor protein because heat treatment to denature protein structure abolished speciﬁc binding of [3H]NMS and the high-afﬁnity component of [3H]dimethyl-W84 binding (Fig. 6A, inset). To check for the speciﬁcity of saturable binding, other putative ligands for the allosteric site, i.e., alcuronium, W84, and gallamine, were applied. They displaced [3H]dimethyl-W84 binding at concentrations that matched the concentrations for the allosteric effect of these agents on NMS dissociation. The linear correlation between the pEC50,diss values and the binding constants pK of the four applied modulators [pEC50,diss ¼ 0.87 pK þ 0.90, r2 ¼ 1.00 (Fig. 6B)] was excellent; thus, high-afﬁnity [3H]dimethyl-W84 binding apparently occurs at the site that mediates the allosteric effect of the modulators on NMS–receptor complexes. The low-afﬁnity component of [3H]dimethyl-W84 binding (Fig. 6A) probably represents nonspeciﬁc, yet saturable, binding of the radioligand to the cardiac membranes. Since there is evidence that modulators have fast binding kinetics at their site of action (8,81), in this initial study, membranes were separated in the [3H]dimethyl-W84 binding experiments by centrifugation instead of ﬁltration with subsequent washing (10). A 20,000-fold higher binding afﬁnity at the allosteric site of M2 receptors compared with the nonspeciﬁc binding afﬁnity

Figure 6 (Figure on facing page) (A) Inhibition of [3H]dimethyl-W84 total binding (0.3 nM) in porcine cardiac membranes by nonlabeled dimethyl-W84. Means SEM of seven experiments performed as quadruplicate determinations are given. The curve is based on a two-site model. Horizontal dashed line: nonsaturable nonspeciﬁc binding. Inset: Effect of increasing concentrations of dimethyl-W84 on the binding of [3H]dimethyl-W84 to M2 receptors occupied by 1 mM NMS after preincubation of the membranes at 80 C for 15 minutes in a water bath. Ordinate: [3H]Dimethyl-W84 total binding as a percentage of the value in the absence of nonlabeled dimethyl-W84. Logistic curve ﬁtting. Means SEM of seven experiments, which were performed as quadruplicate determinations, are given. (B) Correlation between the inhibition of [3H]dimethyl-W84 binding by the applied allosteric agents and their inhibitory action on the dissociation of [3H]NMS. Ordinate: pKD, (log)-equilibrium dissociation constant of [3H]dimethyl-W84 binding. pKI, (log)-equilibrium inhibition constant for the inhibition of [3H]dimethyl-W84 binding by the indicated modulators—both determined at NMS (1 mM)–occupied M2 receptors. pEC50, (log) concentration of the modulators reducing the rate constant k1 of [3H]NMS dissociation to 50% the control value. Abbreviation: NMS, N-methylscopolamine. Source: From Ref. 10.

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demonstrated that occupation of the allosteric site by [3H]dimethyl-W84 is a preferential event and not an aspect of a nonspeciﬁc interaction with membrane interphases (10). Thus, [3H]dimethyl-W84 proved to be a radiolabeled allosteric modulator of muscarinic receptors with high allosteric potency and exempliﬁed that binding of allosteric agents to the common site can be measured directly in genuine NMS-occupied M2 receptors of cardiac tissue. USE OF THE RADIOALLOSTER [3H]DIMETHYL-W84 TO TEST PREDICTIONS OF THE COOPERATIVITY MODEL FOR THE BINDING OF ALLOSTERIC MODULATORS AT THE COMMON ALLOSTERIC BINDING SITE OF M2 RECEPTORS After having characterized the binding of the new allosteric radioligand, the question arose of whether the ternary complex model of allosteric interactions stands ﬁrm in that its predictions for a given allosteric interaction were independent of whether an allosteric or an orthosteric radioligand was applied as a probe. Until the introduction of the radioalloster [3H]dimethylW84, the receptor binding characteristics of an allosteric agent could not be measured directly, but were deduced from the effects induced by the allosteric compound on the binding of an orthosteric radioligand. The effect on the equilibrium binding of the orthosteric radioligand was analyzed by means of the ternary complex model of allosteric interactions, the so-called ‘‘cooperativity model,’’ which was introduced into the muscarinic ﬁeld by Stockton et al. (11), simpliﬁed by Ehlert (12), and further developed by Lazareno and Birdsall (8) to cover a number of different experimental situations. The cooperativity factor a represents the alloster-induced KD shift of the orthosteric ligand. The cooperativity model predicts that a is reciprocal in nature, i.e., a equals the KA shift of the allosteric agent induced by the orthosteric ligand and vice versa. Until the development of the radioalloster [3H]dimethyl-W84 for a labeling of the common allosteric site in muscarinic acetylcholine M2 receptors (10), this prediction could not be tested by direct binding measurements at the allosteric site. Using [3H]dimethyl-W84 and the centrifugation procedure, we aimed to ﬁnd out whether the KA shift of the allosteric modulator induced by the orthosteric agent occurred as predicted by the cooperativity model (102). We studied the interaction of the prototype allosteric modulator gallamine with the orthosteric ligand NMS because it shows a strong negative cooperativity with NMS. First, we determined the effect of gallamine on the binding of the orthosteric ligand [3H]NMS in the indirect ‘‘conventional’’ way by an analysis of the data according to the cooperativity model. Indeed, we observed a pronounced negative cooperativity between gallamine and NMS as reﬂected by the cooperativity factor a ¼ 47. Second, applying the radioalloster [3H]dimethyl-W84, we directly measured in competititon experiments the Ki of gallamine binding at the allosteric site either in the absence or in the presence of NMS (at a concentration saturating

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Figure 7 Binding characteristics of gallamine at free and at NMS-occupied M2 receptors determined either indirectly by applying [3H]NMS and the ternary complex model of allosteric interactions or directly by competitition experiments with the radioalloster [3H]dimethyl-W84. From [3H]NMS experiments are depicted pKA, (log)-equilibrium dissociation constant of gallamine in free M2 receptors; a, factor of cooperativity between gallamine and NMS. According to the ‘‘cooperativity model’’ the equilibrium dissociation constant of gallamine binding at NMS-occupied receptors is equivalent to p(a KA). From [3H]dimethyl-W84 experiments are depicted pKi, (log)-equilibrium dissociation constant for the competitive inhibition of speciﬁc [3H]dimethyl-W84 binding by gallamine in free and NMS-occupied receptors. Abbreviation: NMS, N-methylscopolamine. Source: From Ref. 102.

the orthosteric site). According to the cooperativity model, the NMS-induced shift of the Ki of gallamine binding should match the cooperativity factor a. Figure 7 compiles in the form of a bar graph the binding characteristics of gallamine in free and NMS-occupied M2 receptors as derived from the experiments with [3H]NMS and [3H]dimethyl-W84, respectively. The binding constants of gallamine for the interaction with free and with NMS-occupied M2 receptors were independent of whether the orthosteric or the allosteric site was radiolabeled. Accordingly, the measured value of the NMS-induced shift of gallamine binding afﬁnity was identical with the shift predicted by the cooperativity model (difference in bar height in Fig. 7). In the meantime a routine ﬁltration procedure for measurement of [3H]dimethyl-W84 binding in free M2 receptors was developed (see below), which has an improved signal to noise ratio allowing analysis of the interaction of [3H]dimethyl-W84 with orthosteric ligands analogously to that of

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[3H]NMS with allosteric agents, i.e., indirectly by means of the cooperativity model. Hereby, we found very similar a values (0.19, 0.27) for the interaction of dimethyl-W84 and NMS, independent of whether [3H]dimethyl-W84 or [3H]NMS was the probe (103). These results further support the ternary complex model of allosteric interactions. Taken together, to determine the binding characteristics of allosteric agents in G-protein–coupled receptors, radioligands are commonly applied that bind to the orthosteric receptor site. Using the radioactive modulator [3H]dimethyl-W84 to directly measure alloster binding at muscarinic M2 receptors, the validity of the ternary complex model of allosteric interactions could be demonstrated. INTERACTIONS OF ALLOSTERIC AND ORTHOSTERIC LIGANDS WITH [3H]DIMETHYL-W84 AT THE COMMON ALLOSTERIC SITE OF MUSCARINIC M2 RECEPTORS The next step in the development of [3H]dimethyl-W84 was to simplify the method used to measure radioalloster binding so that its application within the scope of any imaginable question would become possible. The centrifugation assay used in the initial studies suffers from the limitation of a poor ratio of speciﬁc to nonspeciﬁc binding (0.3:1) caused, in part, by the presence of a saturable nonspeciﬁc [3H]dimethyl-W84 component with an apparent KD of ca. 30 mM (10). This binding component impedes measurements of the afﬁnity of allosteric agents of lower potency. Futhermore, it makes it impossible to obtain detailed information regarding the measurement of cooperativities between [3H]dimethyl-W84 and orthosteric ligands and on whether allosteric ligands might be interacting cooperatively with [3H]dimethyl-W84. Additionally, the time needed for separating the radiolabeled membranes by centrifugation prevented attempts to measure the kinetics of [3H]dimethyl-W84 binding and to determine how they might be affected by orthosteric and other allosteric ligands. Therefore, a more robust [3H]dimethyl-W84 ﬁltration binding assay was developed in which the lowafﬁnity binding component was absent (Fig. 8) and was applied to explore the aforementioned allosteric phenomena, which could not be studied previously. Although the buffer conditions were changed (Table 3), the log afﬁnity constant for [3H]dimethyl-W84 (10) measured with the new ﬁltration assay was close to that found in the centrifugation assay (8.89) (10). Using this assay the interaction of a number of allosteric and orthosteric ligands with the binding of [3H]dimethyl-W84 was investigated (103). In contrast to the centrifugation procedure, which allows measuring of [3H]dimethyl-W84 binding in free and in NMS-occupied M2 receptors, the ﬁltration procedure has until now been successfully applied to monitor [3H]dimethyl-W84 binding in free M2 and M4 receptors (see below). At M1, M3, and M5 receptors the rather low binding afﬁnity of dimethyl-W84

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Figure 8 Heterologous inhibition of speciﬁc [3H]dimethyl-W84 binding (1.5 nM) by the indicated (A) modulators or (B) orthosteric agents at free human M2 receptors in membranes of Chinese hamster ovary cells by applying a ﬁltration binding assay. Ordinate: Speciﬁc [3H]dimethyl-W84 binding as a percentage of control binding in the absence of nonlabeled ligand. Nonspeciﬁc binding was deﬁned in the presence of 10 mM gallamine, The incubation time amounted to 120 minutes at 23 C. The curve ﬁts were based in (A) on logistic curve ﬁtting and in (B) on the ternary complex model of allosteric interactions [for ()-levetimide only logistic curve ﬁtting was carried out]. The data are mean values SEM of two to seven experiments carried out in duplicate or triplicate. Source: From Ref. 103.

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Table 3 Conditions and Characteristics of [3H]Dimethyl-W84 Binding Assays Centrifugation binding assay (10) Assay volume Assay conditions

Recommended radioligand concentration Separation of bound and free

Filter

Washing

Ratio of speciﬁc vs. nonspeciﬁc binding Saturable nonspeciﬁc binding Native membranes processable (e.g., from porcine hearts) Membranes from cultured cells processable (e.g., CHO, COS-7) Measurement of [3H]dimethyl-W84 binding in free M receptors Measurement of [3H]dimethyl-W84 binding in NMSoccupied M receptors Expected pKDa

Filtration binding assay (103)

1.5 mL 0.3 mL 4 mM Na2HPO4, 1 mM 10 mM Hepes; 20 mM NaCl, KH2PO4, pH 7.4 at 23 C and 0.01% BSA, pH 7.4 at for 2 hr 23 C for 2 hr M2: 0.3 nM M2: 1.5 nM, M4: 12 nM

Pelleting by means of a table-top centrifuge, 20 min at ~21,000 g, manual removal of the supernatant, 1.5-mL reaction tubes —

1 1 mL incubation buffer (5 C) (pellet surface and reaction tube walls) ~0.3:1

Filtration by means of a Tomtech Harvester (e.g., Mach III or Mach III M, Wallac1), 96 place 1.2-mL deep well microtiter plates Filtermat A (Wallac), glass ﬁber ﬁltermats pretreated with 0.2% polyethyleneimine 1 0.8 mL 100 mM NaCl (5 C) Up to 4:1

Yes (50–60% of total binding) Yes

No

Yes

Yes, recommended

Yes

Yes

Yes

Not yet demonstrated

M2,NMS occupied: 8.74 (10), M2: 8.89 (10)

M2: 8.47 (103), M4: 6.93

Not recommended

(Continued )

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Table 3 Conditions and Characteristics of [3H]Dimethyl-W84 Binding Assays (Continued ) Centrifugation binding assay (10) Measurement of the afﬁnity of highafﬁnity allosteric modulators Measurement of the afﬁnity of low-afﬁnity allosteric modulators Measurement of the kinetics of [3H]dimethyl-W84 binding Measurement of the afﬁnity of orthosteric agents and their cooperativity with [3H]dimethyl-W84

Filtration binding assay (103)

Yes

Yes

No

Yes

No

Yes

Not recommended

Yes, recommended

a

pKD: negative log value of the equilibrium dissociation constant for the binding of dimethylW84 to the respective muscarinic receptor subtype, either free or occupied by 1 mM NMS. Abbreviations: NMS, N-methylscopolamine; CHO, Chinese hamster ovary; BSA, bovine serum albumin; Hepes, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid.

(cf. Table 2) impedes direct binding measurements with the radiolabeled modulator. A summary of the conditions and characteristics of both [3H]dimethyl-W84 binding assays, which indicates their individual advantages and disadvantages, is given in Table 3. It was possible to demonstrate that several allosteric ligands including gallamine and alcuronium could completely inhibit the speciﬁc binding of [3H]dimethyl-W84 with slope factors not signiﬁcantly different from one (Fig. 8A), which was compatible with an interaction at a single site. A number of orthosteric ligands inhibited the binding of [3H]dimethyl-W84 submaximally (Fig. 8B); an allosteric analysis provided estimates of the afﬁnity of the orthosteric ligands for the unliganded receptor and the cooperativity between [3H]dimethyl-W84 and these ligands. AF-DX 384 differs from a ‘‘pure’’ orthosteric ligand since it seems to interfere with the binding to both the orthosteric site and the common allosteric site of the M2 receptor (62). AF-DX 384 did not fully inhibit the binding of [3H]dimethyl-W84 (Fig. 8B), compatible with an allosteric mechanism in which both ligands can bind simultaneously to the M2 receptor and interact with each other allosterically. If AF-DX 384 solely interacts with the allosteric site as proposed for the analog, AF-DX 116 (104), the interaction between

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[3H]dimethyl-W84 and AF-DX 384 would have been expected to be competitive. [3H]dimethyl-W84 allowed detection of the well-known, very large (10,000-fold) stereoselectivity of dexetimide and levetimide, as well as the relatively low negative cooperativity between dimethyl-W84 and both NMS and atropine. Another group of muscarinic allosteric agents exists that have been termed ‘‘atypical’’ allosteric modulators. It is important to note that a clinically favorable elevation of ligand binding, e.g., of the endogenous agonist acetylcholine, can be achieved by both ‘‘typical’’ (93) and ‘‘atypical’’ allosteric modulators (84). Dimethyl-W84 belongs to the larger group of typical modulators, tacrine and Duo3 (Fig. 1) are examples of the group of atypical allosteric agents. The term ‘‘atypical’’ originally derives from uncommon allosteric actions of these modulators on the binding of orthosteric radioligands characterized by steep curves (27,48,81,97,103,105,106) and a weak propensity to be antagonized by allosteric antagonists like obidoxime (81). An advantage of atypical over typical modulators is the rather small dependence of their allosteric actions on the ionic composition of the receptor surrounding (55). Atypical modulators may therefore provide attractive leads for the development of new allosteric agents with improved properties compared to typical modulators. The modulation of the binding of orthosteric ligands characterized by slope factors greater than one may be interpreted by simultaneous and positively cooperative binding of these ligands to both the orthosteric and the allosteric sites. Both tacrine (48) and Duo3 (Fig. 1) (97) have been reported to inhibit the speciﬁc binding of [3H]NMS with slope factors greater than one. However, tacrine and Duo3 also inhibit the rate constant k1 of [3H]NMS dissociation with slope factors greater than one (47,48,59,81). In dissociation experiments, the orthosteric site is occupied by [3H]NMS and therefore the effects causing the slope factors to be greater than one are undoubtedly allosteric in nature and independent of the occupancy of the orthosteric site. It was found with [3H]NMS that tacrine, Duo3 (103), and a series of allosteric pentacyclic carbazolones (106), in spite of slope factors greater than one in equilibrium and kinetic assays, also behaved as predicted according to the equations for the allosteric ternary complex model with a slope factor inserted. Using [3H]dimethyl-W84 the effects of the atypical allosteric agents, Duo3, and tacrine on the equilibrium binding and dissociation of the radioalloster from M2 receptors have been investigated. It was hypothesized that if these atypical allosteric agents used a second independent allosteric site they might leave [3H]dimethyl-W84 binding unaffected but might affect its rate of dissociation. It was found that the Duo3 and tacrine inhibition curves had slope factors greater than one with potencies very similar to those found for the [3H]NMS inhibition curves (Fig. 8A). Duo3 and tacrine decreased the afﬁnity of [3H]dimethyl-W84 without affecting its Bmax (103) value. Interestingly, [3H]dimethyl-W84 binding to the common allosteric site sensed the

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same effects of Duo3 and tacrine as [3H]NMS binding to, or dissociating from, the orthosteric site (103). Thus, the atypical modulators affected not only the equilibrium binding of [3H]dimethyl-W84 but also in an atypical way. Applying [3H]dimethyl-W84 allowed for the ﬁrst time determination of direct estimates of the kinetic constants of an allosteric muscarinic ligand. The kinetics of [3H]dimethyl-W84 were fast (Fig. 9). Indirect measurements

Figure 9 [3H]dimethyl-W84 binding kinetics at free human M2 receptors in membranes from Chinese hamster ovary cells. (A) Association of [3H]dimethyl-W84. Ordinate: Equilibrium [3H]dimethyl-W84 binding as a percentage of nonspeciﬁc [3H]dimethyl-W84 binding determined in the presence of 10 mM gallamine (lower horizontal dashed line). The mean level of [3H]dimethyl-W84 binding of all measurements is indicated by the upper horizontal dashed line. Abscissa: Time after addition of [3H]dimethyl-W84 (1.5 nM) to the membranes. Data are mean values SEM of two experiments carried out in duplicate. (B) Dissociation of [3H]dimethyl-W84. Ordinate: Binding of [3H]dimethyl-W84 (1.5 nM) as a percentage of the binding level before the start of the dissociation reaction. Abscissa: Time after addition of gallamine (10 mM) to visualize the dissociation of [3H]dimethyl-W84. (C) Dissociation of [3H]dimethyl-W84 in the absence and presence of Duo3 and tacrine. The curves were obtained by monoexponential curve ﬁtting. Data are mean values SEM of two (B) and three to seven (C) experiments performed in duplicate. Source: From Ref. 103.

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that had been performed earlier had only suggested that, independently of an occupancy of the orthosteric site, the allosteric ligand equilibrated very fast (81). Thus, it was no surprise that the association was essentially complete at the ﬁrst time point (15 seconds, Fig. 9A). The association data were compatible with a simple bimolecular association of [3H]dimethyl-W84 with the M2 receptor. The associaton half-life time of [3H]dimethyl-W84 was very fast and was calculated to be ﬁve seconds (corresponding to kþ1 ¼ 2 107 M1 sec1). The dissociation of [3H]dimethyl-W84 could be ﬁtted with a monoexponential function; the estimate of the dissociation half-time was nine seconds (k1 ¼ 0.08 per second, Fig. 9B). The pKD for [3H]dimethyl-W84 derived from the kinetic constants matched that obtained in equilibrium binding experiments (103). It was not possible to detect an effect of tacrine or Duo3 on the dissociation rate of [3H]dimethyl-W84, which could have been interpreted with an action at an allosteric site different from the common allosteric site (Fig. 9C). Though [3H]dimethyl-W84 did not allow the molecular mechanism of action of Duo3 or tacrine to be solved, it revealed that the positive homotropic cooperativity of these agents can also be monitored from the allosteric site. Considering the fact that two allosteric sites on muscarinic receptors have been described (81,84,85) with allosteric ligands, such as gallamine and KT5720, interacting with neutral cooperativity, it is conceivable that Duo3 or tacrine can bind to both these sites but with positive cooperativity. Besides studies focusing on typical and atypical allosteric ligands, [3H]dimethyl-W84 may be useful in future studies to elucidate the mechanism of action of biomolecules that have been suggested to interact noncompetitively with cardiac M2 receptors. Some pathophysiological factors have been postulated to affect M2 receptors allosterically. The ﬁrst is the major basic protein (MBP), which is believed to cause M2 receptor dysfunction and enhance vagally mediated bronchoconstriction in asthma. Eosinophil peroxidase (EPO) is another cationic protein stored less plentifully in eosinophil granules than MBP. Both compounds are allosteric antagonists affecting [3H]NMS binding to and retarding [3H]NMS dissociation from M2 receptors in the upper micromolar concentration range; they do not affect M3 receptors (107,108). The effects of MBP and EPO on M2 receptor airway function can be blocked by polycationic compounds like heparin and poly-L-glutamic acid. Therefore, MBP and EPO may affect M2 function by an electrostatic interaction with negatively charged amino acids on the extracellular face of the receptor protein. In patients with Chagas’ disease, circulating autoantibodies against the second extracellular loop (e2) of the human heart M2 mAChRs have been found to stimulate myocardial M2 receptors in an ‘‘agonist-like’’ fashion (109,110). The activating effect of the autoantibodies at this receptor was blunted by atropine but neutralized by an exogenously administered antipeptide corresponding to the e2 loop of the M2 receptor. An antipeptide

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autoantibody also displayed agonist-like activity (109). In [3H]NMS binding experiments, immunoglobulin G (IgG) fractions from sera of chagasic patients bound noncompetitively to M2 receptors, whereas IgG fractions of healthy blood donors had no effect (111). Moreover, various opioid peptides are known to show a number of nonopioid effects (112), e.g., dynorphin A causes hyperalgesia through the Nmethyl-D-aspartic acid (NMDA) receptor (113). In muscarinic receptors dynorphin inhibits the binding of the muscarinic ligand [3H]NMS at equilibrium and alters the kinetics of ligand dissociation in an allosteric fashion (114). Taken together, the [3H]dimethyl-W84 ﬁltration assay allows screening of various kinds of ligands for an afﬁnity to the allosteric site. This facilitates the testing of the speciﬁcity of allosteric actions. With [3H]dimethylW84 even those agents can be tested that do not alter the equilibrium binding of orthosteric ligands, i.e., show neutral cooperativity. This offers a decisive advantage compared with binding studies performed with orthosteric ligands; their use in screening studies may leave allosterically acting ligands undetected (7). Future [3H]dimethyl-W84 experiments may include the testing of orthosteric agonists, the sensitivity of [3H]dimethyl-W84 binding toward guanine nucleotides, and of selected biomolecules as potential ligands of the allosteric site of muscarinic M2 receptors. Binding of [3H]Dimethyl-W84 to an Allosteric Site in Muscarinic M4 Receptors Applying the ﬁltration binding assay [3H]dimethyl-W84 has also been used to label an allosteric site in muscarinic M4 receptors. [3H]dimethyl-W84 binding was inhibited by the archetypal modulator gallamine in a concentration-dependent manner. The log binding constant calculated for gallamine was pKi ¼ 5.68. Aiming to check whether the inhibition of [3H]dimethylW84 binding by gallamine was speciﬁc, we measured its allosteric interaction with the conventional antagonist [3H]NMS. Allosteric analysis using the ternary complex model yielded a pKA ¼ 6.03 for gallamine binding to free M4 receptors and a sevenfold negative cooperativity between gallamine and NMS. The close correspondence of the measures of afﬁnity for gallamine binding at free M4 receptors measured either with [3H]NMS (pKA ¼ 6.03) or with [3H]dimethyl-W84 (pKi ¼ 5.68) suggests that [3H]dimethyl-W84 speciﬁcally labels the allosteric site on muscarinic M4 receptors (115). COMMON SITE RECEPTOR EPITOPES IDENTIFIED BY SITEDIRECTED MUTAGENESIS AS A BASIS FOR A M2 RECEPTOR PHARMACOPHORE MODEL COMPRISING DIMETHYL-W84 Muscarinic acetylcholine receptors belong to the family of the rhodopsinlike G-protein–coupled receptors, which consist of seven hydrophobic

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transmembrane helices connected by intracellular and extracellular loops, an extracellular amino terminus, and a cytoplasmatic carboxyl terminus. The binding site for the orthosteric ligand acetylcholine is buried in a binding pocket formed by the transmembrane helices (116,117); in contrast, the allosteric binding site is located at the entrance of the ligand binding pocket (43,54,118). Several studies have revealed that the second outer loop and the junction of the third outer loop and the top of transmembrane segment 7 (TM7) contain residues that are important in the binding of muscarinic allosteric ligands. The binding of gallamine is considerably affected by the EDGE (Glu-Asp-Gly-Glu) sequence, that is, the residues 172–175 in M2 (118,119). The subtype selectivities of gallamine and alcuronium are affected by the asparagine residue at M2419 (119,120). The elimination of a tryptophane residue that is conserved among all ﬁve muscarinic receptor subtypes (located at position 422 in M2) reduced the afﬁnity of gallamine and other allosteric ligands (121). Whereas the M2–M5 subtype selectivity of structurally different muscarinic antagonists is determined by different receptor epitopes (122), two regions of the receptor (i.e., o2 and o3) are crucial—though with varying relative importance—to the binding or selectivity of many typical muscarinic allosteric ligands (89). A large number of these compounds appear to interact competitively at a common allosteric site (45,53,81,89), which is likely to reﬂect the close proximity of the residues involved, either near the middle of o2 or near the o3–TM7 junction. As mentioned previously, muscarinic allosteric ligands exhibit generally the highest afﬁnity to the M2 receptor subtype. Multiple studies were directed to identify receptor epitopes that are important for the binding of allosteric agents to the M2 receptor (118,123,124). M2–M5 chimeric receptors have been shown to be helpful to gain more insight into receptor parts that confer the high M2 receptor afﬁnity of allosteric ligands (43,89,119, 123,124). Mainly, these studies focused on the formation of ternary complexes that are the characteristic feature of allosteric interactions by measuring the interaction of the allosteric agents in receptors in which the orthosteric site was occupied by [3H]NMS. Binding of the allosteric agents to the receptor is reﬂected by an inhibition of [3H]NMS dissociation. Applying this procedure, it was possible to identify two epitopes that account fully for the high afﬁnity of alkane-bisammonium–type and caracurine V–type allosteric ligands (Fig. 1) to the M2 receptor, relative to the M5 receptor. One of these epitopes consists of a single amino acid at the beginning of the seventh transmembrane region of the M2 receptor, M2423threonine (Thr) (123); the other is M2177tyrosine (Tyr) in the middle region of the second outer loop o2 (124). These two amino acids are sufﬁcient to completely account for the M2–M5 selectivity of a number of allosteric agents, including dimethyl-84 (Fig. 10), in the NMS-occupied M2 receptor as demonstrated by a double point mutated receptor (M2177Y ! Q, M2423T ! H). It should be mentioned that M2423threonine corresponds to M4436serine. Mutation

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Figure 10 Concentration–effect curves for the allosteric delay of [3H]NMS dissociation induced by dimethyl-W84 at the indicated wild-type and mutant receptors. The receptors were prelabeled with [3H]NMS, dissociation was then induced by addition of 1 mM atropine and observed in the absence or presence of the allosteric modulator. Ordinate: Apparent rate constant k1 of [3H]NMS dissociation as a percentage of control in the absence of modulator. Abscissa: Log concentration of allosteric modulator. Experiments were performed with membranes from transiently transfected COS-7 cells in 5 mM Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 C). Means SEM of two to four separate experiments are given. Nonlinear regression analysis was performed applying a four-parameter logistic function. Abbreviation: [3H]NMS, radiolabeled N-methylscopolamine. Source: From Ref. 124.

of M4436S ! H resulted in a loss of M4–M5 selectivity similar to the loss of M2–M5 selectivity seen with dimethyl-W84 in the M2423T ! H mutant (123). This points to an analogous orientation of dimethyl-W84 in the M4 receptor compared to the M2 receptor protein. It remains to be investigated whether atypical modulators that do not seem to interact competitively with gallamine and the others (81,84,85) may bind to regions other than o2 and o3. There is evidence suggesting that the M2–M5 selectivity of the atypical muscarinic allosteric agents Duo3 is affected by epitopes in o1 and the N-terminal areas of the M2 receptor (125). Equilibrium binding experiments with [3H]NMS and diallylcaracurine V suggest that the amino acids, M2177Tyr and M2423Thr, are also involved in the binding afﬁnity of the allosteric modulators for the free M2 receptor, but additional epitopes appear to contribute to the M2–M5 selectivity under this condition. Based on these ﬁndings a three-dimensional model of the M2 receptor in the NMS-occupied state was generated using the crystal structure of bovine rhodopsin as a template, which seemed to be suitable to gain insight into the putative topography and the molecular mechanisms of allosteric–orthosteric interactions. Placing NMS into the orthosteric site prevents the allosteric agent from utilizing elements of the orthosteric site and

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Figure 11 (See color insert) Position of NMS and dimethyl-W84 in the wild-type M2 receptor. Abbreviations: EDGE, Glu-Asp-Gly-Glu; TM, transmembrane; NMS, N-methylscopolamine. Source: K. Jo¨hren and H. D. Ho¨ltje, Institute of Pharmaceutical & Medicinal Chemistry, University of Du¨sseldorf, Germany, unpublished.

stabilizes an inactive conformation of the receptor protein. It was found that the model gave a hypothesis of how the alkane-bisammonium–type agent W84 and diallylcaracurine V, although structurally diverse, could interact with the same two amino acids in a meaningful fashion (124). The docking of dimethyl-W84 into this M2 receptor model is shown in Figure 11. In summary, the amino acid M2177tyrosine in o2 together with 423 M2 threonine at the top of TM7 accounts entirely for the M2–M5 selectivity of alkane-bisammonium–type allosteric modulators like dimethylW84 at NMS-occupied muscarinic receptors and partially in free receptors. The experimentally observed phenomena could be accommodated in a three-dimensional model of the NMS-occupied M2 mAChR, which provided insight into the topography of ligand binding and the molecular mechanisms of allosteric–orthosteric interactions. CONCLUDING REMARKS The development of the radioligand [3H]dimethyl-W84 has allowed us to plumb the depth of the allosteric principle in muscarinic M2 receptors and

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has broadened our view of the features inherent in this subtype. The interaction with the allosteric site not only confers a high binding afﬁnity to dimethyl-W84 but also contributes to its pronounced M2–M5 selectivity above that of other M2 selective antagonists. [3H]dimethyl-W84 was shown to label an allosteric site used by a number of structurally diverse allosteric modulators that is topologically distinct from the orthosteric neurotransmitter site for acetylcholine. [3H]dimethyl-W84 binding fulﬁlled the basics of receptor theory: its binding occurred with high afﬁnity in a speciﬁc, saturable, reversible, concentration-dependent manner and according to the law of mass action. The routine ﬁltration assay presented for the measurement of [3H]dimethyl-W84 binding should facilitate the monitoring and screening of allosteric actions at M2 and M4 receptors in future studies. Because [3H]dimethyl-W84 binding occurs in accordance with the ternary complex model of allosteric interactions, it may also be used as a probe to determine in structure–activity relationship studies the afﬁnity of orthosteric agents and their cooperativity with [3H]dimethyl-W84 at the allosteric site. The knowledge obtained using dimethyl-W84 in muscarinic receptors and the perspectives opened by it should encourage us to continue the exploitation of allosterism in G-protein–coupled receptors as a challenging but clinically promising principle of pharmacological action. ACKNOWLEDGMENTS The development of [3H]dimethyl-W84 and related studies was supported by the Deutsche Forschungsgemeinschaft (Tr 372/1-1, Ho 1368/7-2, Mo 821/ 1-3) and the Medical Research Council. REFERENCES 1. Christopoulos A, Kenakin T. G protein-coupled receptor allosterism and complexing. Pharmacol Rev 2002; 54(2):323–374. 2. Tucˇek S, Prosˇka J. Allosteric modulation of muscarinic acetylcholine receptors. Trends Pharmacol Sci 1995; 16:205–212. 3. Birdsall NJ, Lazareno S, Matsui H. Allosteric regulation of muscarinic receptors. Prog Brain Res 1996; 109:147–151. 4. Ellis J. Allosteric binding sites on muscarinic receptors. Drug Dev Res 1997; 40:193–204. 5. Christopoulos A, Lanzafame A, Mitchelson F. Allosteric interactions at muscarinic cholinoceptors. Clin Exp Pharmacol Physiol 1998; 25:185–194. 6. Holzgrabe U, Mohr K. Allosteric modulators of ligand binding to muscarinic acetylcholine receptors. Drug Discov Today 1998; 3:214–222. 7. Christopoulos A. Allosteric binding sites on cell-surface receptors: novel targets for drug discovery. Nat Rev Drug Discov 2002; 1(3):198–210. 8. Lazareno S, Birdsall NJM. Detection, and veriﬁcation of allosteric interactions of agents with labeled and unlabeled ligands at G-protein-coupled

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13 a2-Adrenoceptors Physiology, Pharmacology, and Allosteric Modulation Emma S. J. Robinson Department of Pharmacology, School of Medical Sciences, University Walk, University of Bristol, Bristol, U.K.

Alan L. Hudson Psychopharmacology Unit, University of Bristol, Bristol, U.K. and Department of Pharmacology, Medical Sciences Building, University of Alberta, Edmonton, Canada

INTRODUCTION The adrenoceptors belong to the large family of seven transmembrane (TM) G-protein–linked receptors. The very fact that adrenoceptors are coupled to G-proteins is the primary mechanism for allosteric interaction that results in intracellular changes to elicit a biological response. They are cell membrane receptors and mediate the effects of the endogenous catecholamines, adrenaline, and noradrenaline. The adrenoceptor family has been subdivided into three pharmacologically and functionally distinct subgroups. The major subgroups of adrenoceptor are now classiﬁed as the a1-adrenoceptors, a2adrenoceptors and b-adrenoceptors (1–3). The subdivision of the a-adrenoceptors was initially based on anatomical location and the discovery of prejunctional autoreceptors, which regulate the release of noradrenaline. Langer (4) proposed that a-adrenoceptors in the sympathetic nervous system could be subdivided into a1-adrenoceptors, located postsynaptically, 327

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and a2-adrenoceptors, which were located presynaptically and regulated the release of noradrenaline through a negative feedback system. However, the pharmacological characterization of the a-adrenoceptor subtypes resulted in the identiﬁcation of the ‘‘presynaptic’’ a2-adrenoceptor in a postsynaptic location and, thus, a1- and a2-adrenoceptors were characterized pharmacologically rather than anatomically (5). The a2-adrenoceptors are the target for several different therapeutic agents currently licensed for use in human and veterinary medicine (Table 1). In particular, a2-adrenoceptors are known to function both as autoreceptors, regulating the release of noradrenaline, and as heteroreceptors, regulating the release of other neurotransmitter substances and therefore provide a mechanism through which these transmitters can be manipulated. This may explain why they are involved in a broad array Table 1 Functional Roles Mediated by a2-Adrenoceptors in the Central Nervous System, Their Proposed Mechanism of Action, and Therapeutic Implications Disease target Depression

Cognition

Schizophrenia

Neurodegeneration

Pain

Hypertension Drug withdrawal Anesthesia

Proposed mechanism of action Presynaptic inhibition of monoamine transmitter release mediated by auto and heteroreceptors Stimulation of neurotrophic factors such as BDNF Increased noradrenergic innervations and decreased presynaptic inhibition of cholinergic pathways Unclear

Clinically relevant compounds Mirtazapine Mianserin Tricyclic antidepressants None at present

Clozapine (a2-AR antagonist plus other actions) Increased dopamine neurotransmission Idazoxan mediated through reduced pre-synaptic Efaroxan inhbition and enhanced noradrenergic Fipamezole innervation hydrochloride Increased release of neuroprotective neurotrophic factors Enhanced descending inhibitory Amytrityline pathways Clonidine Adjunct to opioid analgesics Moxonidine Decreased sympathetic outﬂow Clonidine Decreased CNS stimulation Clonidine Decreased LC ﬁring Lofexidine Decreased LC ﬁring Xylazine Metetomidine

Abbreviations: BDNF, brainderived neurotrophic factor; CNS, central nervous system; LC, locus coeruleus; AR, adrenoceptor.

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of physiological functions, which include regulation of blood pressure, regulation of insulin release, sedation, analgesia, and thermogenesis, and have a critical role in behaviors such as cognition, mood, and sleep. At the cellular level the a2-adrenoceptor is a G-protein–coupled receptor (GPCR), which primarily leads to inhibition of adenylyl cyclase, activation of Kþ channels, and inhibition of voltage-sensitive Ca2þ channels.

SUBTYPES OF a2-ADRENOCEPTORS The development of new methods for investigating receptors such as molecular biological techniques has resulted in the identiﬁcation of four a2-adrenoceptors with distinct binding proﬁles and amino acid sequences, the a2A-, a2B-, a2C-, and a2D-subtype (6). Subsequent analysis suggests that the a2A- and a2D-adrenoceptors are species homologues of the same subtype that display a distinct pharmacological proﬁle. Therefore, each species expresses three different a2-adrenoceptors with the a2A/D- and a2Csubtypes primarily expressed in the central nervous system (CNS) and all three subtypes found in the peripheral nervous system and on postsynaptic tissues (6). The lack of highly selective ligands for the different a2-adrenoceptor subtypes has meant that characterization of the functional roles mediated by the different subtypes has relied on alternative techniques such as genetically modiﬁed animals. The three different a2-adrenoceptor subtypes identiﬁed in mice have now been studied using mice expressing a mutant, knocked-out or overexpressed form of the receptor of interest. In addition, there have been extensive studies using antisense oligonucleotides to investigate a2-adrenoceptor subtype speciﬁc functions. A summary of the results from functional studies characterizing the subtype speciﬁc functions are given in Table 2. Studies like these are now revealing the diversity of functions associated with these receptors and potential applications for a2-adrenoceptor agonists and antagonists and the potential for allosteric modulation. Furthermore, advances in imaging a2-adrenoceptors alongside studies into subtype-speciﬁc functions have led to renewed interest in the application of a2-adrenoceptor ligands to treat neurological and psychiatric disorders.

a2-ADRENOCEPTOR LOCALIZATION AND SUBTYPE DISTRIBUTION IN THE CNS a2-Adrenoceptors are found in both the central and peripheral nervous system in pre- and postsynaptic locations. In the CNS, receptor autoradiography has been used to localize a2-adrenoceptors to speciﬁc brain areas. Utilizing the 10–20-fold selectivity of [3H]rauwolscine and [3H]MK912 with a displacing ligand such as oxymetazoline, which has a higher afﬁnity for the a2A-subtype over the a2C-subtype, the distribution of the a2C-adrenoceptors in the rat brain has been determined (7,8). The highest levels of

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Table 2 Radioligand Binding, Molecular Biological and Functional Evidence for the Different Subtypes of the a2-Adrenoceptor Available at Present Receptor subtype Gene location Rat Human Selective agonists Non-subtype selective agonists Selective antagonists Non-subtype selective antagonists Radioligands Physiological function

Transduction mechanism

a2A/DRG20 (a2D) a2-C10 (a2A) Guanfacine, oxymetazolinea UK14,304,

a2BRNG a2-C2 None

a2CRG10 a2-C4 None

clonidine, p-aminoclonidine

ARC239, imiloxan, Rauwolscinea, prazosin, MK912a spiroxatrine Yohimbine, RX821002, idazoxan, RS15385, rauwolscine, RS79948, SKF86466, MK912 BRL44408

[3H]RX821002, [3H]yohimbine, [3H]rauwolscinea, [3H]MK912a, [3H]UK14,304, [3H]RS79948 197 Hypotension, Vasoconstriction, ? sedation, pressor response analgesia, anesthesia, inhibition of neurotransmitter release Inhibition of adenylyl cyclase via Gi/o activation, #cAMP, inhibition of voltage-gated Ca2þ channels, activation of Ca2þ-dependent Kþ channels

a

Denotes partial selectivity for one a2-adrenoceptor subtype. Abbreviation: cAMP, cyclic adenosine monophosphate.

a2C-adrenoceptors are detected in the striatum, the caudate putamen, and globus pallidus, while areas such as the frontal cortex and locus coeruleus (LC) appear to be predominantly the a2A-subtype (7,8). There has been no report of [3H]prazosin binding in the CNS; thus, the presence of the a2B-subtype has not been demonstrated using this method. The lack of highly selective ligands for a2-adrenoceptor subtypes, however, has meant that subtype speciﬁc localization studies have primarily relied on alternative techniques such as in situ hybridization and immunohistochemistry. The distribution of the messenger RNA (mRNA) coding of these receptors can be achieved using in situ hybridization studies (9–12), although this only provides information of the cells’ ability to produce the receptor and is not a direct measure of the receptor itself.

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The a2A-adrenoceptor mRNA is widely distributed throughout the CNS and is detected in areas such as the cerebral cortex, hypothalamus, and dorsal root ganglia and is exclusively detected in the septum and the locus coeruleus. The a2C-adrenoceptor mRNA also has a relatively widespread distribution in the CNS and is found in high levels in areas such as the cerebral cortex, hippocampus, olfactory bulb, and dorsal root ganglia. The a2CmRNA is also exclusively expressed in the striatum and cerebellar cortex. In contrast to the relatively diverse localization of a2A- and a2C- mRNA, the a2B-adrenoceptor mRNA has been detected exclusively in the thalamus. Immunohistochemistry and immunocytochemistry provide an alternative approach to characterizing the localization of receptor subtypes. Distribution of both a2A- and a2C-adrenoceptor immunoreactivity has been reported in rat CNS (13,14). Co-localization experiments have also been employed to localize a2A- or a2C-adrenoceptors with speciﬁc neuronal markers. For example, two different studies have used co-localization protocols to investigate a2C-adrenoceptors populations on catecholaminergic and gamma- aminobutyric acid (GABA)-ergic neurons in brain stem and midbrain or striatum (15–18). a2A-Adrenoceptor immunoreactivity was observed in areas where the a2A-mRNA had previously been reported and, in general, the autoradiography, in situ hybridization studies, and immunocytochemical studies yield a similar distribution. The a2A-adrenoceptor appears to predominate in the brainstem and areas associated with cardiovascular function, in the prefrontal cortex, and cortex, and in the dorsal horn of the spinal cord (13,19). a2A-Adrenoceptors are also located in the lower brainstem on both catecholaminergic and serotonergic neurons innervating the spinal cord (20). Interestingly, however, immunoreactivity was also seen in the cerebellar cortex and striatum; both these areas have previously been shown to express high levels of the a2C-mRNA (13,19). The distribution of a2C-immunoreactivity is consistent with that seen in in situ hybridization studies (14). The presence of a2C-immunoreactivity in the midbrain associated with dopaminergic regions has been identiﬁed, suggesting the a2C-adrenoceptor may be involved in modulation of dopaminergic function (16). There have been no reports, using antibodies, of the distribution of a2B-adrenoceptors in the CNS despite successful measurement of this subtype in the periphery (21). The subcellular distribution of the a2-adrenoceptors has also been examined to localize the receptor proteins within individual neurons or glial cells (15,17,22). a2-ADRENOCEPTOR-MEDIATED FUNCTIONS IN THE CNS The a2-adrenoceptors have been shown to mediate a diverse range of functions both centrally and peripherally (2). For example, the a2-adrenoceptor agonist clonidine has been shown to produce bradycardia, hypotension, hypothermia, sedation, inhibition of motor function, mydriasis, and

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antinociception in animals (2,23,24). The functional roles mediated by the different a2-adrenoceptor subtypes have been enhanced by developments in genetic approaches such as genetically modiﬁed animals and antisense technology. The ﬁrst of these studies was performed by Link et al. (25) and MacMillan et al. (26) and revealed the differential effects of each a2adrenoceptor subtype on cardiovascular parameters. These studies showed that the a2A- and a2B-adrenoceptor subtypes were, respectively, involved in the hypotensive and pressor response observed with a2-adrenoceptor agonists in vivo; however, manipulation of the a2C-adrenoceptor had no effect on cardiovascular parameters. This led to the conclusion that agonists selective for the a2C-adrenoceptor over the a2A- and a2B-subtypes would lack cardiovascular side effects. Following these experiments, many different groups have utilized targeted genetic approaches to determine the potential applications associated with the different a2-adrenoceptor subtypes relative to the known effects of a2-agonists and antagonists (27,28). Details of the proposed functional roles mediated by the a2A- and a2C-adrenoceptor subtypes are given in Tables 3 and 4, respectively. The most studied function associated with a2-adrenoceptors is regulation of transmitter release (42). a2-Adrenoceptors mediate an inhibitory effect of transmitter release associated with their coupling to Gi/o-proteins, activation of which results in reduced Ca2þ inﬂux and enhanced Kþ efﬂux. a2-Autoreceptors localized on noradrenergic cell bodies reduce cell-ﬁring rates, while a2-autoreceptors located at the presynaptic terminal inhibit transmitter release. A second category of presynaptic a2-adrenoceptors, termed heteroreceptors, are found on the presynaptic terminal of neurons containing neurotransmitters other than noradrenaline (Fig. 1). Therefore, a2-adrenoceptor agonists attenuate noradrenaline release via stimulation of both presynaptic and somatodendritic a2-adrenoceptors. Conversely, a2-adrenoceptor antagonists increase basal noradrenaline levels through blockade of these receptors (43–47). Interestingly, the effects observed with a2-antagonists in vivo suggest basal noradrenaline levels are under tonic control by a2-autoreceptors. The ﬁrst a2-heteroreceptor was localized to cholinergic neurons, stimulation of which inhibits acetylcholine release in rat, guinea pig, and human brain (42,48,49). a2-Adrenoceptors have also been identiﬁed on 5-hydroxytryptamine (5-HT) neurons where they inhibit the release of 5-HT and are thus designated a2-heteroreceptors (50). In vivo dialysis studies have shown that the a2-adrenoceptor antagonist, idazoxan, can block the reduction in 5-HT induced by clonidine, but does not facilitate 5-HT release when administered alone (51). Although a2-adrenoceptor antagonists do not appear to facilitate 5-HT release increasing noradrenaline levels by administration of desipramine, a noradrenaline uptake blocker, reduces 5-HTinduced attenuation of hippocampal CA3 pyramidal cell activity in the rat (52). Acute and chronic administration of idazoxan has also been shown

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Table 3 Summary of the Functional Roles Mediated by a2A-Adrenoceptors Characterized Using Genetically Modiﬁed Mice or Antisense Oligonucleotides Method used Functional knockout mouse (D79N)

Knockout mouse

Antisense

Functional effect

Reference

Attenuated hypotensive response to agonist

MacMillan et al. (26)

Attenuated hypothermic, antinociceptive and sedative response to agonist Impaired working memory Protective role in models of anxiety and depression Reduced inhibitory response to endogenous and exogenous receptor stimulation Increased NA metabolite in cortex and thalamus/ hypothalamus Increased anxiety, locomotor activity, heart rate and piloerection Impaired motor coordination Increased basal systolic blood pressure Attenuated hypnotic response to an agonist Attenuated behavioral and hypothermic response to an agonist Increased behavioral activity Increased corticosterone and reduced anxiety

Hunter et al. (29)

Franowicz et al. (30) Schramm et al. (31) Trendelenberg et al. (32)

La¨ndesma¨ki et al. (28)

Nunes, (33) Mizobe et al. (34) Robinson et al. (35)

Robinson et al. (36) Shishkina et al. (37)

Abbreviation: NA, noradrenaline.

to enhance the ﬁring rate of the dorsal raphe 5-HT neurons (53). This effect is most likely due to enhanced noradrenergic innervations of the raphe nucleus activating postsynaptic a1-adrenoceptors that increase the ﬁring rate. A similar mechanism is also hypothesized for noradrenergic innervations of the substantia nigra and therefore dopaminergic projections to the basal ganglia. Direct measurement of dopamine levels in the brain using in vivo microdialysis revealed a small increase in striatal dopamine following a2-adrenoceptor antagonist administration (54,55) and, in mouse striatum, a2-adrenoceptor agonists have been reported to decrease dopamine

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Table 4 Summary of the Functional Roles Mediated by a2C-Adrenoceptors Characterized Using Genetically Modiﬁed Mice or Antisense Oligonucleotide Method used Knockout or overexpression

Antisense

Functional effect

Reference

Dopamine turnover in the striatum

Sallinen et al. (38)

Locomotor response to d-amphetamine Cognitive function Regulation of cortical EEG arousal Minor regulatory role in inhibiting response to endogenous and exogenous agonist Inhibitory role in processing sensory information. Regulation of motor and emotion-related behaviors Reduced inhibition of cAMP accumulation in striatum

Sallinen et al. (38) Bjorklund et al. (39) Puolivali et al. (40) Trendelenberg et al. (32) Scheinin et al. (27)

Lu and Ordway (41)

Abbreviations: EEG, electroencephalogram; cAMP, cyclic adenosine monophosphate.

release during physiological stimulation frequencies (56). Although the evidence suggests an inhibitory role for the a2-adrenoceptor in dopamine neurotransmission, the location of these receptors and mechanism have still to be fully elucidated. The a2C-adrenoceptor subtype has been reported to predominate in the striatum and nigrostriatal pathway and may be involved directly or indirectly in regulation of dopamine neurotransmission (38,57,58). Immunohistochemical studies have shown that at least a proportion of the a2C-adrenoceptors located in the striatum are localized on medium spiny GABAergic neurons; therefore, activation of these receptors functions to directly regulate the release of GABA rather than dopamine. Studies in other brain areas support these ﬁndings and suggest a2-heteroreceptors are expressed on certain GABAergic neurons. Data obtained from basal forebrain GABAergic neurons suggest the expression of an a2A-adrenoceptor subtype (59) and, an indirect effect on histaminergic neurons in the hypothalamus resulting from a2-adrenoceptor-mediated presynaptic inhibition of GABAergic inputs (60). In terms of amino acid transmitters, a2-heteroreceptors may also regulate glutamate release in certain brain areas. Functional studies have shown that glutamatergic transmission in the hippocampus (61,62), amygdala (63), and spinal cord (64,65) are regulated by a2-adrenoceptors. The experiments performed in the hippocampus identiﬁed the subtype responsible for the effects observed as the a2A-adrenoceptor (61). Finally, the potential role for drugs that manipulate noradrenergic function should also be considered in relation to regulation of endogenous

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Figure 1 Diagrammatic representation of a2-adrenoceptor-mediated regulation of neurotransmission. Somatodendritic autoreceptors and presynaptic autoreceptors regulate NA release. The combined regulation of NA release and subsequent activation of postsynaptic a1-adrenoceptors and presynaptic heteroreceptors regulate the release of DA and 5-HT. In the basal ganglia, a2-adrenoceptors are also located on GABAergic neurons while hippocampul a2-adrenoceptors have been located on glutamatergic neurons. The functional effects associated with regulation of these amino acid transmitters are still in the early stages of investigation. Studies using genetic techniques and immunohistochemical localization studies may elucidate further potential targets for a2-adrenoceptor ligands, in particular the potential for a2-adrenoceptor subtype speciﬁc ligands. Abbreviations: LC, locus coeruleus; NA, noradrenaline; DA, dopamine; 5-HT, 5-hydroxytryptamine; GABA, gamma-aminobutyric acid.

neurotrophic factors. Noradrenergic innervations from the locus coeruleus are proposed to contain and release neurotrophic factors; thus, a2-adrenoceptors, through their ability to regulate noradrenergic function, provide a mechanism through which neurotrophic factors such as brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF) can be regulated (66,67). a2-ADRENOCEPTORS IN NEUROLOGICAL AND PSYCHIATRIC DISORDERS The ability of a2-adrenoceptors to regulate the release of noradrenaline and other neurotransmitter substances in the brain has resulted in a number of different therapeutic agents whose primary site or action is stimulation or inhibition of a2-adrenoceptors. In terms of the CNS, the major disease targets are depression and neurodegenerative and cognitive diseases such as Parkinson’s disease and Alzheimer’s disease. a2-Adrenoceptor agonists are also licensed for the treatment of drug withdrawal and provide useful adjunct therapy in chronic pain management (Table 2). Finally, modulation of noradrenaline may

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underlie the effects seen with drugs like amphetamine in the treatment of attentional disorders such as attention deﬁcit hyperactivity disorder. Depression The role of the a2-adrenoceptors in the regulation of neurotransmitter release is of particular interest within the ﬁeld of depression and anxiety-related disorders. Noradrenaline and serotonin are the two major neurotransmitters implicated in depressive disorders, and elevating brain levels of these neurotransmitters is associated with improved mood and motivation. The ability of a2-adrenoceptor antagonists to enhance noradrenergic neurotransmission and the changes that occur in a2-adrenoceptors in depression suggest that these receptors may play an important role in the etiology and treatment of depression (68). For example, delays in the onset of action of antidepressant drugs have been shown to correlate with the time course of down regulation of certain monoamine receptor populations. In particular, all antidepressant treatments, including electroconvulsive shock therapy, are associated with a down regulation in a2-adrenoceptor expression and function in the CNS (68,69). The a2-adrenoceptors are further implicated in a study where evidence from depressed suicide victims suggests an up regulation in a2A-adrenoceptor subtype (7,70). The ability of drugs that act at a2-adrenoceptors to function as antidepressant drugs has been clearly shown by the success of the a2-adrenoceptor and 5-HT receptor antagonist, mirtazapine. This drug binds to a2-adrenoceptors in the LC to increase noradrenaline release in higher brain centers such as the hippocampus and frontal cortex. Enhanced noradrenergic stimulation of raphe neurons also results in an increase in 5-HT release. These effects are further enhanced by blockade of presynaptic a2-autoreceptors and heteroreceptors (Fig. 1). The role of noradrenaline may also exceed beyond the current monoamine hypothesis of depression, where an increase in synaptic concentrations of noradrenaline and/or serotonin are proposed to underlie the improvements in mood and motivation observed. The role of noradrenaline in the control of neurotrophic factors such as BDNF has led to the hypothesis that stimulation of these factors may be the route through which antidepressant efﬁcacy is achieved. For example, increased noradrenergic neurotransmission and subsequent release of neurotrophic factors may reduce apoptotic rate and increase neurogenesis in the hippocampus (71) resulting in an improvement in mood. Neurodegenerative and Cognitive Diseases Noradrenaline is believed to play a signiﬁcant role in the etiology of neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s

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disease (67). Manipulation of the noradrenergic system through a2-adrenoceptors or by some form of allosteric modulation provides an opportunity to address the symptoms and progression of these diseases at several levels. 1. Deﬁcits in dopaminergic and cholinergic function can be modiﬁed through blockade of presynaptic heteroreceptors. 2. Stimulation of noradrenergic innervations from the locus coeruleus. 3. Noradrenergic-mediated stimulation of neurotrophic factors. The LC-noradrenergic system shows signiﬁcant reductions in the number of neurons (>80%) innervating dopaminergic regions such as the substantia nigra and ventral tegmental area in postmortem tissue from Parkinson’s disease patients (72). This deﬁcit is likely to inﬂuence the function of nigrostriatal dopamine systems and stimulation of noradrenergic function through a2-adrenoceptor blockade and may provide an alternative approach to treating Parkinson’s disease (54,67). A similar view is also held in relation to the progressive neurodegenerative disorder, Alzheimer’s (67). Although a detailed understanding of the etiology of this disease has not been established, noradrenaline may regulate cortical cholinergic transmission. In both of these diseases, blockade of a2-adrenoceptors with selective antagonists results in a disinhibition of the LC-noradrenergic system and presynaptic receptors (73,74). These effects result in increased transmitter release, which can counteract the deﬁcits resulting from the neurodegenerative disease. In addition, stimulation of LC neurons increases the release of cotransmitter and co-factor substances, such as BDNF (75), ﬁbroblast growth factor (76), and adenosine triphosphate (ATP) (77), which confer protective and trophic properties. These facts have led to the hypothesis that a2-adrenoceptor antagonists may reduce the symptoms and progression of neurodegenerative diseases. Fipamezole hydrochloride is a non-subtype selective a2-adrenoceptor antagonist that is currently undergoing phase II clinical trials for Parkinson’s disease (78). a2-Adrenoceptors are also thought to play an important role in cognitive function. There appears to be a facilitatory action of both a2-adrenoceptor agonists and antagonists on memory function, which is most likely due to agonist stimulation of postsynaptic receptors and the ability of a2adrenoceptor antagonists to elevate central noradrenaline levels, which acts on postsynaptic receptors in the prefrontal cortex (79–81). This hypothesis was demonstrated in monkeys where very low doses of yohimbine, acting via an indirect mechanism, and clonidine, via a direct mechanism, facilitate memory function (79). Studies in genetically modiﬁed animals suggest that the a2A- and a2C-adrenoceptors may mediate opposing actions in relation to cognitive function. Mice overexpressing the a2C-adrenoceptor show a deﬁcit in spatial working memory that can be reversed by treatment with the a2-adrenoceptor antagonist, atipamizole (39). Studies in the D79N, a2Aadrenoceptor knockout mouse also revealed a memory impairment (30),

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but relating to a deﬁcit rather than overexpression of the speciﬁc subtype. In contrast, a recent study in a2A-adrenoceptor knockout mice has shown that a2-adrenoceptor agonist-induced impairment of memory function is mediated by the a2A-subtype (82). These data have provided an insight into the complexity of a2-adrenoceptor–mediated regulation of cognitive function and highlight the need for further investigation into how different subtypes of a2-adrenoceptors and their respective localization combine to modulate different aspects of cognition. A regulatory role for noradrenaline on the activity of septal cholinergic neurons has also been shown in mice and facilitation of memory function may be due to inhibitory a2-adrenoceptors on cholinergic neurons (83). a2-Adrenoceptors in the basal ganglia have also been proposed to enhance memory consolidation and reinforcement (84). The a2A- and a2Cadrenoceptors are expressed within the basal ganglia and overexpression of the a2C-adrenoceptor has been shown to attenuate spatial working memory in mice, leading to the conclusion that a2C-adrenoceptor antagonists may enhance cognitive function at this level (39). The ability of a2-adrenoceptor ligands to improve cognitive function has led to the proposal that these compounds may have therapeutic beneﬁts in dementia and the treatment of diseases such as Parkinson’s disease, schizophrenia, and frontal lobe dysfunction (67,80,85,86). Overall, understanding of the regulation of cognition through a2A- and a2C-adrenoceptors is in its infancy but preliminary ﬁndings indicate opposing actions for a2A- and a2C-subtypes in different brain regions. As a consequence, subtype-speciﬁc ligands may provide a mechanism for more selective manipulation applicable to the individual disease etiology. a2-ADRENOCEPTORS AND ALLOSTERIC INTERACTIONS There are several reported allosteric interactions with a2-adrenoceptors, namely coupling to G-proteins, which is of primary importance, modulation of ligand binding by Naþ and Hþ ions, and inhibition of antagonist binding by amiloride. G-Protein Modulation The a2-adrenoceptor belongs to the superfamily of GPCRs and possesses seven domains that span the cell membrane separating the internal machinery of the cell from the external environment. The binding of an agonist to the a2-adrenoceptor results in a physiological effect because of an interaction with G-protein(s) to cause an intracellular event in the form of a signaling cascade. The binding of an agonist to the a2-adrenoceptor on the external surface of the cell results in a conformational change that causes coupling to a G-protein. In common with other GPCRs, a2-adrenoceptors are linked to more than one G-protein. Primarily, a2-adrenoceptors mediate

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inhibition of adenylyl cyclase by binding to Gi/o-proteins which results in inhibition of cyclic adenosine monophosphate (cAMP) production (Table 2). In contrast, coupling to Gs-proteins stimulates adenylyl cyclase activity increasing the production of cAMP (87). Of course the Gi/o subfamily of G-proteins has nine a-subunits, which allows for signiﬁcant functional diversity. This simple allosteric interaction, as shown throughout this volume, does not reﬂect the true state of affairs. Allosteric is the Greek word for ‘‘other site’’ and is a term coined by Monod et al. (88) for the application of enzyme kinetics. It was then adapted to relate to cell surface receptor function (89). The limitation of this model is that it only accounts for two interconvertible states for the receptor, namely active and inactive. Therefore, this simple allosteric model has been replaced by a ternary complex model that allows for multistate complexes of the receptor and a G-protein (90). Such a model allows for allosteric transition states and can explain agonist efﬁcacy and inverse agonism (91). Furthermore, for a2-adrenoceptors there are now results to support Kenakin’s (92) idea for agonist directed trafﬁcking (93) (see following text). Of the seven TM domains that make up the a2-adrenoceptor it is thought that the extracellular areas on TM3, TM5, and TM6 are important for ligand binding (Fig. 2) with particular emphasis on TM5 (94,95). The intracellular ends of TM3 and TM6 domains are important for G-protein coupling; of these cytoplasmic extensions, the TM6 helix has been shown to be important for Gi/o activation (96), whereas the helix of TM5 is involved in Gs activation (97). It has also been shown that catecholamines bind to the extracellular side of TM5. More speciﬁcally, the hydroxyl groups on the phenyl ring of catecholamines interact with Ser204 of the TM5 domain and contribute to receptor activation (98). Recent data support the role of Ser204 and also point to Ser200 having a role in agonist activity (94). In other studies it has been demonstrated the Cys351 is required for UK14,304 to have full agonist activity and mutation to Gly351 resulted in partial agonist activity for UK14,304 (99). In terms of the subtypes of a2-adrenoceptors there may be a differential binding of G-proteins. In a variety of cell lines the a2A-adrenoceptor can couple to both Gi/o- and Gs-proteins (96,97). Similarly, for the a2Badrenoceptor it is coupled to Gi/o-proteins to inhibit adenylyl cyclase (87) and in PC12 cells the a2B-adrenoceptor is linked to stimulation of cAMP production via Gs-proteins (100). The a2C-adrenoceptor can also bind Gi/oproteins and to a lesser extent may bind Gs-proteins (101). Prolonged exposure to an a2-adrenoceptor agonist results in receptor desensitization due to phosphorylation by G-protein–coupled receptor kinases (GRKs) followed by arrestin binding (102). There is evidence, at least in HEK239 cells, that prolonged agonist exposure leads to internalization of a2B-adrenoceptors but not a2A-adrenoceptors (103); whether this is the case in other cell lines or takes place in vivo remains to be seen.

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Figure 2 Schematic representation of the human a2A-adrenoceptor showing the seven TM domains, proposed ligand binding site (TM5), critical sites for phosphorylation (Ser296,297,298,299), and possible docking area for G-proteins (Gi/o). Abbreviations: TM, transmembrane; ARK, adrenoceptor kinase. Source: From Ref. 24.

Co-transfection studies in COS-7 cells have provided good evidence that the GRKs responsible for the phosphorylation are bARK and bARK2, which are known to phosphorylate b-adrenoceptors (97). This GRK-mediated phosphorylation leads to the uncoupling of the receptor from the G-protein (104) and is speciﬁc to particular amino acids, namely the serines and threonines of the third intracellular loop (Fig. 2). These amino acids are phosphorylated, and this leads to rapid desensitization (105) in the case of a2A- and a2B- adrenoceptors, less so for a2C-adrenoceptors (106). These studies have been carried out in cell lines expressing the speciﬁc receptor subtypes and for the human a2A-adrenoceptor site-directed mutagenesis studies have shown that serine residues 296, 297, 298, and 299 in the third intracellular loop are phosphorylated (Fig. 2) and all four must be phosphorylated for desensitization to occur (107). Further work by Liggett’s group has found that agonist structure but not intrinsic activity were related to receptor phosphorylation (108). Their work showed that the a2-adrenoceptor agonist UK14,304 did not cause the same degree of phosphorylation as para-aminoclonidine and they predict it may be possible to design agonists that have

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intrinsic activity but would not cause desensitization (108). Whether this would be of therapeutic use is debatable as the sedation associated with antihypertensive drugs clonidine and moxonidine is a side effect that patients come to tolerate over time. Agonist Directed Trafficking, Protean Agonism and RASSL Receptors Kenakin hypothesized the basis of agonist-directed trafﬁcking of receptor signaling (92 and this volume). This, in simple terms, means that when a receptor signals through two or more pathways there can be great diversity in the signaling and subsequent physiological events. a2-Adrenoceptors signal mainly through Gi/o; however, Gi/o has a subfamily of nine a-subunits that allows for considerable diversity. This has led to the suggestion that different a2-adrenoceptor agonists may favor particular signaling pathways, particularly when compared to the catecholamines (109). In a study by Kukkonen et al. (109) the ability of 19 agonists to elevate Ca2þ and inhibit forskolin-induced cAMP production mediated by a2-adrenoceptors was investigated in HEL 92.1.7 cells. The catecholamines were much less potent in decreasing cAMP than in increasing Ca2þ, whereas the opposite was found for imidazoline ox-/thiazolazepine compounds. They suggested that different agonists preferentially activate particular signaling pathways and that different receptor states have the ability to activate different G-proteins. This differential coupling is also supported by other studies, including a study of porcine a2A-adrenoceptors expressed in CHO-K1 cell lines (93). This study found that a classical b-adrenoceptor agonist, isoproterenol, was able to produce a Gs-selective conformation of the a2A-adrenoceptors, whereas known a2-adrenoceptor agonists produced a2-adrenoceptor– mediated responses through both Gs- and Gi-proteins. Such studies do point to agonist-directed trafﬁcking of receptor signaling and may have implications for future drug development. Another term used to describe novel agonist behavior at GPCRs is ‘‘protean agonism’’ where, depending on the state of the receptor, compounds should act as either an agonist or inverse agonist (92). Such a theory would explain why in HEL 92.1.7 cells expressing a2A-adrenoceptors the enantiomers of medetomidine produced opposing responses (110). Taking the aforementioned ideas further has led to the proposition that mutant GPRCs could be found where the receptor is insensitive to the endogenous ligand and has been termed ‘‘receptors activated solely by synthetic ligands’’ or RASSL receptors (111). For a2-adrenoceptors much work has centered around mutant receptors; as described earlier, the TM domain 5 is thought to be important for binding catecholamines. Elegant experiments by Pauwels et al. (94) have examined mutant Ser200Ala and Ser204 Ala, and wild type a2-adrenoceptors coexpressed fused to Ga15-protein in

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Figure 3 Comparison between a2-adrenoceptor agonist-induced Ca2þ responses as obtained in CHO-K1 cells co-transfected with a Ser200Ala a2A AR and Ga15-protein (upper panel) or Ser204Ala a2A-adrenoceptor and Ga15-protein (lower panel). The response to ()adrenaline is signiﬁcantly attenuated in the cells expressing the Ser204 Ala a2A-adrenoceptor. Ca2þ responses were measured as described by Pauwels and Colpaert and expressed as a percentage of the maximal Ca2þ response induced by UK14,304 (1 mM). Curves were constructed using mean values s.e. mean obtained in 2 to 3 independent transfection experiments. Abbreviations: AR, adrenoceptor. Source: From Ref. 94.

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Table 5 Competition Binding Afﬁnities (Ki) of Agonists vs. Either Agonist Binding ([3H]UK14,304) or Antagonist Binding ([3H]RX821002) to the Wild-Type Human a2A-Adrenoceptor and Receptor Mutants Expressed in CHO Cells

Apparent Ki (nM), vs. [3H]UK14,304 R-Adrenaline R-Noradrenaline S-Noradrenaline Dopamine UK14,304 p-Aminoclonidine Clonidine Apparent Ki (nM) vs. [3H]RX821002 R-Adrenaline R-Noradrenaline Dopamine UK14,304

a2A-wt

a2ASer201

a2ASer201Cys204

1.4 0.2 4.6 1.0 105 23 34 6 0.33 0.09 0.9 0.1 1.5 0.04

3.8 1.1 10.7 0.9 233 38 123 4.7 1.3 0.1 1.1 0.2 1.4 0.2

563 263 2530 330 11,970 1670 4970 236 1.7 0.3 0.5 0.1 0.4 0.1

3240 390 13,900 1,080 43,500 2,400 224 16

57,4000 10,000 152,000 32,800 310,000 35,700 1,395 (n ¼ 2)

59,000 2,000 313,000 41,000 419,000 91,000 1770 95

Source: From Ref. 95.

CHO cells (94). Whereas synthetic ligands medetomidine and clonidine could elicit a Ca2þ response in the mutants, adrenaline lacked intrinsic activity in the Ser204Ala mutant and was a partial agonist at the Ser200Ala a2-adrenoceptors mutant (Fig. 3). These data are backed up by radioligand binding studies performed in CHO cells transfected with wild type human a2A-adrenoceptors or mutants containing Ser201 or Ser201Cys200 or Ser201Cys204; again these residues are in the TM domain 5 (95). When an agonist [3H]UK14,304 was used to label the receptors, catecholamine afﬁnity was signiﬁcantly reduced in the Ser201Cys204 mutant, while the afﬁnity for clonidine actually increased (Table 5). When the antagonist [3H]RX821002 was used to label a2-adrenoceptors, catecholamine afﬁnity was signiﬁcantly reduced for both mutants, moreover, so was afﬁnity for UK14,304 (Table 5). In the same study, agonist-induced [35S]GTPcS binding was carried out, and whereas UK14,304 was a full agonist at the wild type receptor, it was a partial agonist at both mutants (95). This all serves to point out the importance of these conserved serine residues for agonist binding and may also have implications for future drug development (94). IONIC MODULATION AND EFFECTS OF AMILORIDE It has been well established by Limbird’s group that digitonin-solubilized pig brain a2-adrenoceptors can undergo allosteric interaction by ions, notably

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Naþ and Hþ. Sodium chloride, lithium chloride, and to a lesser extent, potassium chloride increased the afﬁnity of [3H]yohimbine for porcine brain a2-adrenoceptors (112). In the same study it was demonstrated that lowering the pH of the assay buffer reduced receptor afﬁnity for the agonist adrenaline. It was also shown that amiloride accelerated the dissociation of [3H]yohimbine from porcine a2-adrenoceptors. The authors concluded that an allosteric binding site was responsible for the changes seen in [3H] yohimbine binding. Further experiments in Limbird’s laboratory expressed a2-adrenoceptors in the COS-M6 cell line and found Naþ increased the afﬁnity of labeled yohimbine binding; lowering the pH or adding 5-amino-substituted amiloride analogues accelerated the dissociation of [3H]yohimbine (113). More recent work has examined a ternary model for the allosteric modulation of a2adrenoceptors by amiloride (114). In this study, the authors examined amiloride interacting with the binding of the agonist [3H]UK14,304 and with agonist induced binding of [35S]GTPcS in CHO cells. Their ﬁndings showed that amiloride and its derivatives decreased the dissociation rate of [3H]UK14,304 from a2-adrenoceptors and had a concentration-dependent decrease on noradrenaline or UK14,304 induced [35S]GTPcS binding. This led to the conclusion that the cooperativity between the agonist and amiloride was independent of receptor–G-protein coupling as the functional assays were performed in the presence of guanosine diphosphate (GDP) (114). FUTURE PROSPECTS OF ALLOSTERIC MODULATION OF a2-ADRENOCEPTORS In terms of a2-adrenoceptors one could suggest that allosteric modulation is in its infancy and there have been some interesting publications with regard to potential drug development. Drugs with true selectivity for the subtypes of a2-adrenoceptors are not yet available, whether it would be possible to have prolonged antihypertensive activity without sedation in an a2-adrenoceptor agonist remains to be seen. Given that a2-adrenoceptor antagonists may have therapeutic use in the treatment of Parkinson’s disease, they may also be able to facilitate memory and cognition, which are certainly valid reasons for searching for ligands that can allosterically modulate a2-adrenoceptors. Allosteric modulation may prove a more subtle and useful way to modulate or provide therapeutic manipulation of a2-adrenoceptor function and provide drugs with fewer side effects and improved therapeutic proﬁle. Finally, as the allosteric interactions differ among the subtypes of a2-adrenoceptors this may provide a mechanism of identifying subtype selective compounds in the future. REFERENCES 1. Minneman KP, Esbenshade TA. Alpha1-adrenergic receptor subtypes. Annu Rev Pharmacol Toxicol 1994; 34:117–133.

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Figure 12.11 (See p. 316)

Position of NMS and dimethyl-W84 in the wild-type M2 receptor.

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