Current Neuropharmacology - Volume 5, Issue 3, 2007

Drugs acting at G-protein coupled receptors (GPCRs) represent the core of modern medicine. Traditionally, these drugs target the orthosteric site of the receptors, i.e. the binding site of the endogenous agonist. An increasing number of patents describe synthetic allosteric modulators of GPCRs, which influence receptor activity at sites distinct from the orthosteric site. This documents that the search for allosteric modulators of GPCRs has become a fully-fledged part of current drug discovery efforts. It is now generally accepted that allosteric modulation of GPCRs holds considerable promise for the development of novel therapeutics, but it has taken decades to persuade the skeptics of the advantages this concept has to offer. Allosteric modulation is now considered a valid strategy to obtain first generation therapeutics as well as second generation alternatives to currently available therapeutics. A key step towards broad exploration of the allosteric concept was the introduction of cell-based functional high-throughput screening assays for GPCRs, which have replaced radioligand binding assays as the primary screen for allosteric compounds. The reviews in this special issue, written by some of the leading scientists in the field, provide an overview of the experimental strategies used to identify and characterize allosteric modulators for Class A, B, and C GPCRs. There are a number of theoretical and practical reasons that support the notion that it is easier to obtain selective and/or effective therapies with allosteric modulators than with conventional competitive ligands. Most importantly, allosteric modulation enables the development of molecular entities that have no equivalent in the world of competitive ligands. With a range of allosteric compounds it is, in principle, possible to stabilize receptors in various biologically active conformations, which allows the chemical adjustment of receptor activity in more sophisticated ways than with orthosteric ligands. It is possible not only to identify allosteric ligands that act as positive or negative allosteric modulators of GPCR function, but also to identify allosteric ligands that act as agonists in their own right. Positive and negative allosteric modulators can affect both affinity and efficacy of the endogenous agonist, thereby significantly expanding the pharmacological repertoire for a given target. Allosteric modulation may sometimes also be the most straightforward way to identify small molecular weight compounds for targets that are already pharmacologically or clinically validated. This is particularly evident for Class B GPCRs, which are activated by peptide ligands and normally display poor chemical tractability. Allosteric modulation therefore offers the opportunity to develop non-peptide therapeutics that can be administered orally and that readily cross the blood-brain barrier. A primary goal in pharmaceutical research is to selectively target novel drugs to defined receptor populations, which is expected to reduce side effects and to expand the range of clinical applications. Given the generally high degree of sequence conservation within orthosteric binding sites of GPCRs, competitive ligands have been very disappointing in terms of their subtype-selectivity. It has been much easier to achieve subtype-selectivity with allosteric ligands that, for example, clearly distinguish members of the metabotropic glutamate receptor or muscarinic receptor families. This supports the concept that the binding sites for allosteric ligands are less conserved than the binding-sites for orthosteric ligands. Because the allosteric and orthosteric sites are conformationally linked, an allosteric modulator could essentially produce a receptor with completely novel reactivities towards the endogenous agonist. Subtype-selective allosteric compounds may therefore additionally impose a degree of functional selectivity to the receptor that contributes to subtype-specific effects. Moreover, since binding of an allosteric ligand can change the interfaces between the receptor and effector molecules, it may be possible to selectively influence a subset of possible signaling pathways. Importantly, from a drug discovery point of view, it is becoming clear that allosteric ligands are less limited in pharmacophore diversity than orthosteric ligands.....

The description of the allosteric modification of receptors to affect changes in their function requires a model that considers the effects of the modulator on both agonist affinity and efficacy. A model is presented which describes changes in affinity in terms of the constant α (ratio of affinity in the presence vs the absence of modulator) and also the constant ξ (ratio of intrinsic efficacy of the agonist in the presence vs absence of modulator). This allows independent effects of both affinity and efficacy and allows the modeling of any change in the dose-response curve to an agonist after treatment with modulator. Examples are given where this type of model can predict effects of modulators that reduce efficacy but actually increase affinity of agonist (i.e. ifenprodil) and also of modulators that block the action of some agonists (the CXCR4 agonist SDF-1α by the antagonist AMD3100) but not others for the same receptor (SDF-1α peptide fragments RSVM and ASLW).

Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a “common” allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR.

Class B GPCR's are activated by peptide ligands, typically 30-40 amino acid residues, that are involved in major physiological functions such as glucose homeostasis (glucagon and glucagon-like peptide 1), calcium homeostasis and bone turnover (parathyroid hormone and calcitonin), and control of the stress axis (corticotropin-releasing factor). Peptide therapeutics have been developed targeting these receptors but development of nonpeptide ligands, enabling oral administration, has proved challenging. Allosteric modulation of these receptors provides a potential route to developing nonpeptide ligands that inhibit, activate, or potentiate activation of these receptors. Here the known mechanisms of allosteric modulators targeting Class B GPCR's are reviewed, particularly nonpeptide antagonists of the corticotropin-releasing factor 1 receptor and allosteric enhancers of the glucagon-like peptide-1 receptor. Also discussed is the potential for antagonist ligands to operate by competitive inhibition of one of the peptide binding sites, analogous to the Charniere mechanism. These mechanisms are then used to discuss potential strategies and management of pharmacological complexity in the future development of allosteric modulators for Class B GPCR's.

The calcium (Ca2+)-sensing receptor (CaR) belongs to family C of the G-protein coupled receptors (GPCRs). The receptor is activated by physiological levels of Ca2+ (and Mg2+) and positively modulated by a range of proteinogenic L-α-amino acids. Recently, several synthetic allosteric modulators of the receptor have been developed, which either act as positive modulators (termed calcimimetics) or negative modulators (termed calcilytics). These ligands do not activate the wild-type receptor directly, but rather shift the concentration- response curves of Ca2+ to the left or right, respectively. Like other family C GPCRs, the CaR contains a large amino-terminal domain and a 7-transmembrane domain. Whereas the endogenous ligands for the receptor, Ca2+, Mg2+ and the L-α-amino acids, bind to the amino-terminal domain, most if not all of the synthetic modulators published so far bind to the 7-transmembrane domain. The most prominent physiological function of the CaR is to maintain the extracellular Ca2+ level in a very tight range via control of secretion of parathyroid hormone (PTH). Influence on e.g. secretion of calcitonin from thyroid C-cells and direct action on the tubule of the kidney also contribute to the control of the extracellular Ca2+ level. This control over PTH and Ca2+ levels is partially lost in patients suffering from primary and secondary hyperparathyroidism. The perspectives in CaR as a therapeutic target have been underlined by the recent approval of the calcimimetic cinacalcet for the treatment of certain forms of primary and secondary hyperparathyroidism. Cinacalcet is the first clinically administered allosteric modulator acting on a GPCR, and thus the compound constitutes an important proof-ofconcept for future development of allosteric modulators on other GPCR drug targets.

The metabotropic glutamate receptor family comprises eight subtypes (mGlu1-8) of G-protein coupled receptors. mGlu receptors have a large extracellular domain which acts as recognition domain for the natural agonist glutamate. In contrast to the ionotropic glutamate receptors which mediate the fast excitatory neurotransmission, mGlu receptors have been shown to play a more modulatory role and have been proposed as alternative targets for pharmacological interventions. The potential use of mGluRs as drug targets for various nervous system pathologies such as anxiety, depression, schizophrenia, pain or Parkinson's disease has triggered an intense search for subtype selective modulators and resulted in the identification of numerous novel pharmacological agents capable to modulate the receptor activity through an interaction at an allosteric site located in the transmembrane domain. The present review presents the most recent developments in the identification and the characterization of allosteric modulators for the mGlu receptors.

γ-aminobutyric acid (GABA) plays important roles in the central nervous system, acting as a neurotransmitter on both ionotropic ligand-gated Cl--channels, and metabotropic G-protein coupled receptors (GPCRs). These two types of receptors called GABAA (and C) and GABAB are the targets of major therapeutic drugs such as the anxiolytic benzodiazepines, and antispastic drug baclofen (lioresal®), respectively. Although the multiplicity of GABAA receptors offer a number of possibilities to discover new and more selective drugs, the molecular characterization of the GABAB receptor revealed a unique, though complex, heterodimeric GPCR. High throughput screening strategies carried out in pharmaceutical industries, helped identifying new compounds positively modulating the activity of the GABAB receptor. These molecules, almost devoid of apparent activity when applied alone, greatly enhance both the potency and efficacy of GABAB agonists. As such, in contrast to baclofen that constantly activates the receptor everywhere in the brain, these positive allosteric modulators induce a large increase in GABAB-mediated responses only WHERE and WHEN physiologically needed. Such compounds are then well adapted to help GABA to activate its GABAB receptors, like benzodiazepines favor GABAA receptor activation. In this review, the way of action of these molecules will be presented in light of our actual knowledge of the activation mechanism of the GABAB receptor. We will then show that, as expected, these molecules have more pronounced in vivo responses and less side effects than pure agonists, offering new potential therapeutic applications for this new class of GABAB ligands.

Addiction involves complex physiological processes, and is characterised not only by broad phenotypic and behavioural traits, but also by ongoing molecular and cellular adaptations. In recent years, increasingly effective and novel techniques have been developed to unravel the molecular implications of addiction. Increasing evidence has supported a contribution of the nuclear transcription factor CREB in the development of addiction, both in contribution to phenotype and expression in brain regions critical to various aspects of drug-seeking behaviour and drug reward. Abstracting from this, models have exploited these data by removing the CREB gene from the developing or developed mouse, to crucially determine its impact upon addiction-related processes. More recent models, however, hold greater promise in unveiling the contribution of CREB to disorders such as addiction.

Prenatal exposure to tobacco smoke is a major risk factor for the newborn, increasing morbidity and even mortality in the neonatal period but also beyond. As nicotine addiction is the factor preventing many women from smoking cessation during pregnancy, nicotine replacement therapy (NRT) has been suggested as a better alternative for the fetus. However, the safety of NRT has not been well documented, and animal studies have in fact pointed to nicotine per se as being responsible for a multitude of these detrimental effects. Nicotine interacts with endogenous acetylcholine receptors in the brain and lung, and exposure during development interferes with normal neurotransmitter function, thus evoking neurodevelopmental abnormalities by disrupting the timing of neurotrophic actions. As exposure to pure nicotine is quite uncommon in pregnant women, very little human data exist aside from the vast literature on prenatal exposure to tobacco smoke. The current review discusses recent findings in humans on effects on the newborn of prenatal exposure to pure nicotine and non-smoke tobacco. It also reviews the neuropharmacological properties of nicotine during gestation and findings in animal experiments that offer explanations on a cellular level for the pathogenesis of such prenatal drug exposure. It is concluded that as findings indicate that functional nAChRs are present very early in neuronal development, and that activation at this stage leads to apoptosis and mitotic abnormalities, a total abstinence from all forms of nicotine should be advised to pregnant women for the entirety of gestation.

Due to an overlook on the author's side, some of the references in the legends of figures of an article L-Glutamate and its ionotropic receptor in the nervous system of cephalopods” by A. Di Cosmo, C. Di Cristo and J. B. Messenger, published in the journal Current Neuropharmacology Volume 4, issue 4, October 2006, were wrong. The complete list of correct references is as follows: Figure 1 Reference 49 Figure 2 Reference 16 Figure 3 Reference 21 Figure 5 Reference 12 Figure 6 Reference 33 Figure 7 Reference 33 Figure 8 Reference 25