As the largest family of membrane-bound signal transduction proteins, G protein-coupled receptors
(GPCRs) have been the focus of extensive research efforts over many years and the subject of thousands
of publications and hundreds of reviews over the past several years alone [1-3]. Also referred to
as G protein-linked receptors, serpentine receptors, heptahelical receptors and seven-transmembrane
domain receptors (7TMRs) because they traverse the cell membrane seven times, their exact numbers
are unknown, though over 1000 members have been identified. They are involved in 80% or more of
the signal transduction processes that occur across cell membranes, exerting their effects primarily
through their association with heterotrimeric guanine nucleotide binding G proteins in response to a
wide array of extracellular ligands, from single photons to neurotransmitters, hormones and peptides.
It is estimated that 40% or more of all marketed drugs act through this receptor family . This is
perhaps not so surprising considering they are present in nearly every organ system in higher species and implicated in a wide
spectrum of physiological processes and thus practically every disease area, including CNS disorders, pain, inflammation, cancer,
cardiovascular disease, diabetes and obesity. Still, the relatively low fraction of known GPCRs currently targeted for drug
intervention suggests that many others remain largely untapped that may yet serve to address currently unmet medical needs.
Like most marketed drugs, those acting through GPCRs generally function by competing with endogenous ligands that bind
naturally at active (orthosteric) sites. In general, such orthosteric based drugs have been discovered through various established
strategies familiar to most readers here. These include, for example, rational structure-based design methods predicated on
privileged core templates and derivatives; fragment-based lead design; serendipitous lead discovery by high-throughput screening
(HTS) of large combinatorial libraries; from synthetic analogs inspired by therapeutic natural products; or through in-silico
modelling techniques. Such strategies, often employed in concert, have garnered success with numerous enzyme and receptor
targets, particularly where the active sites have been accurately mapped by x-ray crystallography or approximated from their
complimentary, endogenous ligands.
In more recent years, however, increasing interest has turned towards the search for small molecule GPCR ligands that bind
at allosteric sites (from the Greek allos, "other" and stereos, "space"). Such allosteric binding can impart topological and free
energy changes to the receptor , including the active site region. This can produce an added dimension of functional complexity,
eliciting a pharmacological response not seen with an orthosteric ligand alone and may be intimately co-dependent on
the ligand, receptor and cell type. Some of the unique and formidable challenges, as well as the therapeutic potential of this
non-traditional approach to drug discovery, have been the subject of several recent reviews [6-11].
The conceptual roots of allosterism can be traced to the seminal work on digestive enzymes conducted by Pardee, Umbarger
and others in the 1950s. They found that certain enzymes involved in metabolic pathways were inhibited by some of the diverse
products produced by downstream enzymes involved in those same pathways [12, 13]. This led to the concept of metabolic
regulation through a “negative feedback mechanism”, subsequently described in the 1960s by Monod and co-workers as allosteric
inhibition [14, 15]. Further research into understanding the biochemistry and pharmacology of these complex catalytic and
regulatory processes have become the focus of increasing interest and scope ever since, including within the field of GPCR
drug discovery (vide supra).
There are many potential rewards to comprehending this multivariate complexity and integrating it into the drug discovery
process. These may include compounds with enhanced affinity, efficacy, or novel modes of action; fine tuning or modulation of
functional response, allowing greater physiologic control and a broader spectrum of activities not possible with an orthosteric
ligand alone; reduced desensitization or tolerance, which can occur with orthosteric ligands due to receptor overstimulation
from continual or high agonist exposure; or enhanced molecular, cell and tissue selectivity with lower adverse or off-target effects.
It may also open new avenues to what have been referred to as “nondruggable GPCRs”, those receptors which appear to
be strongly validated as drug targets but for which medicinal chemistry efforts have failed to produce orthosteric ligands as
marketed drugs or even clinical candidates [16, 17].
Overshadowing these potential rewards, however, are a host of formidable technical and development challenges associated
with an allosteric approach to drug discovery, which often requires very different tactics than some of the traditional approaches
mentioned above for orthosterics. For example, an ostensibly active hit or series of hits may appear promising on first
inspection but often turn out to be chemically intractable on further study, producing complex, nonlinear, or flat SAR profiles.
This divergent or modular SAR landscape may result from the highly anisotropic nature of allosteric sites, as opposed to the
centrality and highly conserved nature of orthosteric sites in receptor and enzyme networks . Cell-based functional or phenotypic
assays are often needed to properly identify and characterize allosteric ligands, as opposed to the standard screening
methods for orthosterics using radioligand competition assays, presenting additional obstacles to progress. Also, since allosteric
ligands generally produce little or no activity on their own, assays are often conducted in conjunction with physiologically relevant
endogenous ligands, the nature and levels of which may profoundly affect the SAR profile or functional response . Notwithstanding these and other hurdles typically encountered along the development path for any drug candidate, several
allosterics have recently reached the market. These include Cinacalcet (Sensipar), a calcium-sensing receptor (CaSR) positive
allosteric modulator (PAM) for the treatment of hyperparathyroidism and hypercalcemia , and Maraviroc, a CCR5 negative
allosteric modulator (NAM) for the treatment of HIV infection [20, 21]. A number of other promising candidates are also in the
pipeline or under development [22, 23].
For some perspective, the earliest examples of synthetic allosteric drugs were the benzodiazepines, discovered serendipitously
in the mid-1950s, the first marketed example being Librium, a GABAA receptor modulator introduced in 1960 for acute
treatment of anxiety and alcohol withdrawal syndrome . Related analogs were approved over the next decade or so, including
diazepam (Valium), nitrazepam (Mogadon), temazepam (Restoril) and flurazepam (Dalmane) to treat a variety of CNS disorders.
Like many of the benzodiazepines, however, Librium is a rather blunt instrument and presents significant abuse potential
leading to tolerance or addiction with use beyond a few weeks or months, earning it a Schedule IV classification for controlled
substances. Clearly, there is need for a deeper and more fundamental understanding of the biochemistry and molecular
biology of allosterism with therapeutically promising and validated GPCR targets, which may ultimately lead to more effective
and safer drugs with novel modes of action.
This special issue on Allosteric Modulators of G Protein-Coupled Receptors presents five articles reviewing previous and
ongoing work towards the identification and development of positive allosteric modulators for targets currently of high interest
in the research community, namely the mGlu2, mGlu4, mGlu5 and M1, receptors, as well as an overview of the emerging field of
GPCR dimers and complexes, an area that has attracted increasing interest in recent years.
In our opening contribution, Kuduk and Beshore  review the rationale and history of targeting M1, a member of the
muscarinic receptor family, for which some agonists have shown promise in animal models and clinical trials for the treatment
of Alzheimer’s Disease and schizophrenia. Their search for more potent and efficacious compounds led to the discovery of
benzyl quinoline carboxylic acid (BQCA) through an HTS program, characterized as a highly selective M1 positive allosteric
modulator. Their SAR optimization strategies and in vivo studies on this and related advanced leads are discussed in detail.
Huang, Doller and co-workers  describe recent developments in both orthosteric and allosteric drug discovery efforts
directed towards mGlu4, a target implicated in a variety of therapeutic areas, including Parkinson’s Disease, multiple sclerosis,
inflammatory pain, ischemia and cancer. The authors review the molecular and cell biology of this highly studied receptor, including
the known signaling pathways in the CNS and elsewhere. The potential role of heterodimers, an area reviewed in depth
in a later article of this issue, and recent progress with radioligands and PET ligands are also discussed. Various strategies for
allosteric drug discovery are suggested, given our current level of understanding for this receptor.
Szabó and Keser  review the classes of compounds and advanced clinical candidates identified as PAMs for mGlu2
over the period 2001-2013, a receptor that has shown promise for the treatment of a number of CNS disorders, including
schizophrenia and anxiety. The broad patent activity and diversity of structural templates being pursued indicate the high level
of interest in this area in recent years at both large and small pharma. The authors discuss many of the advanced leads and SAR
studies helping to identify key pharmacophore elements that could benefit ongoing and future drug discovery efforts.
Hao and Xiong  discuss work published over the past decade directed toward PAMs for mGlu5 with a focus on advanced
compounds under study in vivo. This metabotropic receptor is shown to be strongly linked both physically and functionally
to NMDA receptors, which have been centrally implicated in the NMDA hypofunction hypothesis of schizophrenia and
other CNS disorders. The authors review some of the considerable challenges associated with drug development in this area,
including possible mechanism-based neurotoxicity of mGlu5 PAMs reported by several groups and the inconsistency of some
assays across different institutions. On the other hand, observations by some other groups with lead compounds exhibiting affinity-
or efficacy-modulating effects with PAMs on this receptor provide encouraging prospects that effective drugs with
higher margins of safety and an adequate therapeutic index may emerge in coming years.
Hurevich and Gilon  conclude our issue with a review of work being pursued in their labs and others in the rapidly
emerging field of GPCR dimers, oligomers and clusters. Until fairly recently, this area had received relatively scant attention,
not to mention considerable controversy and even skepticism, primarily due to the lack of adequate or reliable methods to properly
study, characterize or even prove the existence, fleeting or otherwise, of such highly complex and dynamic protein aggregates.
Over the past decade or so, however, improvements in bio-analytical techniques have opened new opportunities for research,
which are gradually providing evidence of important roles such dimers and higher order oligomers play in cell signaling
processes. Some complexes have been linked to inflammatory and immune system functioning, for example, and so may play a
significant role in immune-deficient diseases like HIV and multiple sclerosis, as well as Parkinson’s disease and schizophrenia,
among others. Also discussed are some of the strategies being pursued to identify and synthesize small molecules, peptidomimetics
and cyclic peptides designed to inhibit receptor aggregation through allosteric mechanisms.
The field of allosterism has progressed remarkably since its inception in the mid-20th Century and has gradually come to be
appreciated as often playing an integral role in many enzymatic and receptor processes. The ubiquity of GPCRs in extracellular
signaling virtually assured allosteric ligands would also play a significant role here as well, including their incorporation into
the drug discovery process, as chronicled by numerous publications and reviews in recent years, including this journal as well
[4, 29, 30]. As our database of validated and prospective drug targets continues to expand and mature  and our understanding of GPCR signaling is further refined at the cellular and molecular level , it seems likely that allosteric modulators will
bring additional novel, more effective and safer drug therapies to the table.