Allosteric Receptor Modulation in Drug Targeting, 1st Edition

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iv Preface

focus of interest in allosterism was associated with ionotropic cholino-

ceptors. 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 applica-
tion 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, glutamater-
gic and nicotinic receptors, as well as metabotropic G-protein–coupled
receptors such as mGluR, muscarinic, GABAB, and a-adrenoceptors. How-
ever, 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 Möhler
in section 2 provides up-to-date information on their characteristics. Subse-
quent 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 fea-
tures. 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

© 2006 by Taylor & Francis Group, LLC

 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

© 2006 by Taylor & Francis Group, LLC

vi Contents

3. Predicting Dose–Response Curve Behavior . . . . . . . . . . . . 39

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: IONOTROPIC RECEPTORS

4. Allosteric Modulation of GABAA Receptors . . . . . . . . . . . 79

Hanns Möhler
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
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

© 2006 by Taylor & Francis Group, LLC

Contents vii

Concluding Remarks . . . . 118

References . . . . 119

6. 5-HT3 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

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

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

© 2006 by Taylor & Francis Group, LLC

viii Contents

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 . . . . . . . . . . 235

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

© 2006 by Taylor & Francis Group, LLC

Contents ix

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 Tränkle
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

© 2006 by Taylor & Francis Group, LLC

x Contents

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

© 2006 by Taylor & Francis Group, LLC

 Contributors

D. Bertrand Department of Neuroscience, Centre Médical Universitaire,

Geneva, Switzerland

Stephen Daniels Welsh School of Pharmacy, Cardiff University,

Cardiff, U.K.

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 Médical 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

xi

© 2006 by Taylor & Francis Group, LLC

xii Contributors

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 Möhler 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 Tränkle 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.

© 2006 by Taylor & Francis Group, LLC

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 pathophysio-
logical 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 selectiv-
ity, 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 treat-
ment 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 recep-
tor class. This again offers little opportunity for selectivity and the likelihood
of an unacceptable side effect profile.

© 2006 by Taylor & Francis Group, LLC

2 Daniels

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 ortho-
steric 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 advan-
tages 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 pres-
ence 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 (flu-
mazenil) 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 exem-
plified 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 arrange-
ment 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

© 2006 by Taylor & Francis Group, LLC

Allosteric Modulation of Receptor Function 3

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 interac-
tions than equilibrium binding methods (8).
However, functional assays are far more likely to reliably detect allo-
steric interactions and, with the advent of a variety of different techniques
(reporter systems, yeast and melanophore systems, fluorescence-based intra-
cellular 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 defin-
ing 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 oleamide-
induced 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 speci-
fic 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 pharmaco-
dynamic 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 anxio-
genic and either convulsant or proconvulsant and full antagonists

© 2006 by Taylor & Francis Group, LLC

4 Daniels

(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 depen-
dence. Recent experiments using molecular genetic techniques have begun to
establish the benzodiazepine binding sites and to dissect the GABAA recep-
tor 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 approxi-
mately 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 anti-
convulsant activity of diazepam was tested against pentylenetetrazole-
induced 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 con-
trast, 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

© 2006 by Taylor & Francis Group, LLC