The structure and dynamics of GPCR oligomers: a new focus in models of cell-signaling mechanisms and drug design

G protein-coupled receptors (GPCRs) represent approximately half of the potential pharmaceutical targets for current drugs, and thus the way in which these receptors assemble into dimeric/oligomeric structures is of vital interest in practical as well as conceptual aspects of current drug discovery efforts. The significance of such structures is based on the recent realization that ligand-dependent signaling by GPCRs is not necessarily transduced to the G protein by receptor monomers, but possibly by GPCR dimers or even oligomers that function as dynamic macromolecular assemblies. In addition, recent evidence that GPCR hetero-oligomerization can produce signaling units with unexpected combinations of pharmacological properties suggests entirely new methods for developing successful drugs. The dynamic mechanisms of these signaling assemblies remain to be elucidated. The development of increasingly accurate dynamic molecular models of GPCR dimers is expected to produce a more complete structural context for understanding the molecular mechanisms of GPCR function, and to aid in drug discovery.     

Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators

Areas covered in this review: The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery. Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on 'old' or 'new' GPCR targets are very high. Given the fact that the pace at which new 'GPCR drugs' appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective. Take home message: To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers. What the reader will gain: The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.     

Intercellular Lipid Mediators and GPCR Drug Discovery

G-protein-coupled receptors (GPCR) are the largest superfamily of receptors responsible for signaling between cells and tissues, and because they play important physiological roles in homeostasis, they are major drug targets. New technologies have been developed for the identification of new ligands, new GPCR functions, and for drug discovery purposes. In particular, intercellular lipid mediators, such as, lysophosphatidic acid and sphingosine 1-phosphate have attracted much attention for drug discovery and this has resulted in the development of fingolimod (FTY-720) and AM095. The discovery of new intercellular lipid mediators and their GPCRs are discussed from the perspective of drug development. Lipid GPCRs for lysophospholipids, including lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylcholine, free fatty acids, fatty acid derivatives, and other lipid mediators are reviewed.     

Physiological relevance of GPCR oligomerization and its impact on drug discovery

The potentially large functional and physiological diversity of G-protein coupled receptor (GPCR) dimers has generated a great deal of excitement about the opportunity that dimerization provides for enabling novel drug discovery. The discovery of physiologically relevant GPCR dimers suggests that new drug targets for diseases such as schizophrenia and pre-eclampsia can be developed by targeting dimers. Most of the previous work on GPCR dimers made use of the overexpression of differentially tagged GPCRs in heterologous cell systems. Current emphasis on the development of physiologically relevant cell systems that endogenously express the appropriate combination of GPCR dimers and accessory proteins is leading to dramatic increases in our understanding of GPCR dimers. These and other new tools such as GPCR-specific antibodies will be required to develop GPCR dimer specific drugs. Given that ligands are available for only a small percentage of the large number of potentially druggable GPCRs, the use of GPCR dimers might provide the necessary targets to increase the breadth and depth of receptors available for therapeutic interventions.     

Principles: receptor theory in pharmacology

Pharmacological receptor theory is discussed with special reference to advances made during the past 25 years. Thus, the operational model has supplanted analysis of drug-receptor interaction in functional systems whereas the extended ternary complex model is used routinely to simulate quantitatively G-protein-coupled receptor (GPCR) behavior. Six new behaviors for GPCRs, centered on spontaneous production of receptor active states, ligand-selective receptor active states, oligomerization with other proteins (receptor and non-receptor) and allosteric mechanisms, have been characterized and each holds the potential for new drug discovery for therapeutic benefit.     

Overcoming the Challenges of Detecting GPCR Oligomerization in the Brain

G protein-coupled receptors (GPCRs) constitute the largest group of membrane receptor proteins controlling brain activity. Accordingly, GPCRs are the main target of commercial drugs for most neurological and neuropsychiatric disorders. One of the mechanisms by which GPCRs regulate neuronal function is by homo- and heteromerization, with the establishment of direct protein-protein interactions between the same and different GPCRs. The occurrence of GPCR homo- and heteromers in artificial systems is generally well accepted, but more specific methods are necessary to address GPCR oligomerization in the brain. Here, we revise some of the techniques that have mostly contributed to reveal GPCR oligomers in native tissue, which include immunogold electron microscopy, proximity ligation assay (PLA), resonance energy transfer (RET) between fluorescent ligands and the Amplified Luminescent Proximity Homogeneous Assay (ALPHA). Of note, we use the archetypical GPCR oligomer, the adenosine A_2A receptor (A_2AR)-dopamine D₂ receptor (D₂R) heteromer as an example to illustrate the implementation of these techniques, which can allow visualizing GPCR oligomers in the human brain under normal and pathological conditions. Indeed, GPCR oligomerization may be involved in the pathophysiology of neurological and neuropsychiatric disorders.     

G-protein-coupled receptor heterodimers: pharmacology, function and relevance to drug discovery

The growing recognition that members of the rhodopsin-like family A G-protein-coupled receptors (GPCRs) exist and function as dimers or higher-order oligomers, and that GPCR hetero-dimers and -oligomers are present in physiological tissues, offers novel opportunities for drug discovery. Differential pharmacology, function and regulation of GPCR hetero-dimers and -oligomers suggest means to selectively target GPCRs in different tissues and hint that the mechanism of function of several pharmacological agents might be different in vivo than anticipated from simple ligand-screening programmes that rely on heterologous expression of a single GPCR.     

En route to new blockbuster anti-histamines: surveying the offspring of the expanding histamine receptor family

With the recognition of two new histamine receptors at the start of the new millennium, the field of histamine research has seen a clear revival. In the last 10 years, many academic and industrial groups have taken up the challenge to target these new members of the aminergic G-protein-coupled receptor (GPCR) family. Histamine receptor research nicely illustrates how GPCR research has changed in the post-genomic era. There is a growing understanding of GPCR structure, function and modulation at a molecular level. Emerging concepts such as receptor isoforms, GPCR oligomerization and ligand-biased signaling are all being studied, but their clinical relevance remains to be determined. The histamine H(3) and H(4) drug development programs can help to establish the link between these molecular features and clinical efficacy. Several new anti-histamines are now being tested for diverse clinical applications and are poised to become the next blockbuster drugs targeting histamine receptors.     

Molecular modeling of vasopressin receptor and in silico screening of V1b receptor antagonists

Introduction: G protein-coupled receptors (GPCRs) are integral membrane proteins which contain seven-transmembrane-spanning alpha-helices. GPCR-mediated signaling has been associated with various human diseases, positioning GPCRs as attractive targets in the drug discovery field. Recently, through advances in protein engineering and crystallography, the number of resolved GPCR structures has increased dramatically. This growing availability of GPCR structures has greatly accelerated structure-based drug design (SBDD) and in silico screening for GPCR-targeted drug discovery.

Areas covered: The authors introduce the current status of X-ray crystallography of GPCRs and what has been revealed from the resolved crystal structures. They also review the recent advances in SBDD and in silico screening for GPCR-targeted drug discovery and discuss a docking study, using homology modeling, with the discovery of potent antagonists of the vasopressin 1b receptor.

Expert opinion: Several innovative protein engineering techniques and crystallographic methods have greatly accelerated SBDD, not only for already-resolved GPCRs but also for those structures which remain unclear. These technological advances are expected to enable the determination of GPCR-fragment complexes, making it practical to perform fragment-based drug discovery. This paves the way for a new era of GPCR-targeted drug discovery.     

Allosteric modulation of G protein-coupled receptors: A pharmacological perspective

G protein-coupled receptor (GPCR)-based drug discovery has traditionally focused on targeting the orthosteric site for the endogenous agonist. However, many GPCRs possess allosteric sites that offer enormous potential for greater selectivity in drug action. The complex behaviors ascribed to allosteric ligands also present challenges to those interested in preclinical lead discovery. These challenges include the need to detect and quantify various phenomena when screening for allosteric ligands, such as saturability of effect, probe dependence, differential effects on orthosteric ligand affinity vs. efficacy, system-dependent allosteric agonism, stimulus-bias (functional selectivity), and the potential existence of bitopic (hybrid orthosteric/allosteric) ligands. These issues are also critical when interpreting structure-function studies of allosteric GPCR modulators because mutations in receptor structure, either engineered or naturally occurring, can differentially affect not only modulator affinity, but also the nature, magnitude and direction of the allosteric effect on orthosteric ligand function. The ever-expanding array of allosteric modulators arising from both academic and industrial research also highlights the need for the development of a uniform approach to nomenclature of such compounds.     

G protein-coupled receptor allosterism and complexing

G protein-coupled receptors (GPCRs) represent the largest family of cell-surface receptors. These receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. It is now becoming increasingly recognized, however, that the agonist-bound GPCR can also form ternary complexes with other ligands or "accessory" proteins and display altered binding and/or signaling properties in relation to the binary agonist-receptor complex. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of theoretical advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity. Because of the noncompetitive nature of allosteric phenomena, the detection and quantification of such effects often relies on a combination of equilibrium binding, nonequilibrium kinetic, and functional signaling assays. This review discusses the development and properties of allosteric receptor models for GPCRs and the detection and quantification of allosteric effects. Moreover, we provide an overview of the current knowledge regarding the location of possible allosteric sites on GPCRs and candidate endogenous allosteric modulators. Finally, we discuss the potential for allosteric effects arising from the formation of GPCR oligomers or GPCRs complexed with accessory cellular proteins. It is proposed that the study of allosteric phenomena will become of progressively greater import to the drug discovery process due to the advent of newer and more sensitive GPCR screening technologies.     

Computational methods in drug design: Modeling G protein-coupled receptor monomers, dimers, and oligomers

G protein-coupled receptors (GPCRs) are membrane proteins that serve as very important links through which cellular signal transduction mechanisms are activated. Many vital physiological events such as sensory perception, immune defense, cell communication, chemotaxis, and neurotransmission are mediated by GPCRs. Not surprisingly, GPCRs are major targets for drug development today. Most modeling studies in the GPCR field have focused upon the creation of a model of a single GPCR (ie, a GPCR monomer) based upon the crystal structure of the Class A GPCR, rhodopsin. However, the emerging concept of GPCR dimerization has challenged our notions of the monomeric GPCR as functional unit. Recent work has shown not only that many GPCRs exist as homo- and heterodimers but also that GPCR oligomeric assembly may have important functional roles. This review focuses first on methodology for the creation of monomeric GPCR models. Special emphasis is given to the identification of localized regions where the structure of a GPCR may diverge from that of bovine rhodopsin. The review then focuses on GPCR dimers and oligomers and the bioinformatics methods available for identifying homo- and heterodimer interfaces.     

Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson''s disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.     

GPCR偏向性配体介导的选择性功能

G蛋白偶联受体(GPCR)是当今药物治疗中最有效靶向作用的受体超家族之一,它在人类的正常生理状态和疾病过程中都发挥着极大的功效。近年研究发现,GPCR脱敏作用的调节器β-arrestin,可作为真正的衔接蛋白将信号转导到多重效应途径。β-arrestin介导的信号对生化和功能方面的影响力都不同于传统G蛋白介导的信号。由此发现辨别出的多种G蛋白-偏向配体或β-arrestin偏向配体,不仅是用来研究GPCR信号生化特征的有效工具,还具有被开发成治疗药物的潜力。因此,该文就偏向性配体的特点、作用机制、药理学作用及研究偏向性配体的技术进行综述。     

G protein coupled receptors as drug targets: the role of beta-arrestins

G protein coupled receptors (GPCRs) are extremely important drug targets and the beta-arrestin intracellular scaffolding and adaptor proteins regulate major aspects of their pharmacology. beta-arrestin binding to activated, GPCR kinase (GRK)-phosphorylated receptors has the capacity to terminate G protein coupling, internalize the receptors into clathrin-coated vesicles and establish a secondary signaling complex independent of G protein signaling. These events appear to be differentially regulated by GRK phosphorylation, ubiquitination and potentially beta-arrestin oligomerization, which are likely to be highly receptor and cell-type dependent. The role of beta-arrestins in switching from G-protein dependent to independent signaling places them in a pivotal position to dictate the downstream effects of ligand binding. Consequently, we must appreciate the functioning of these molecules as we strive to discover and optimize new GPCR drug therapies for endocrine, metabolic and immune disorders.     

Modeling the 3D structure of GPCRs: advances and application to drug discovery

G protein-coupled receptors (GPCRs) are membrane-embedded proteins responsible for signal transduction; these receptors are, therefore, among the most important pharmaceutical drug targets. In the absence of X-ray structures, there have been numerous attempts to model the three-dimensional (3D) structure of GPCRs. In this review, the current status of GPCR modeling is evaluated, highlighting recent progress made in rhodopsin-based homology modeling and de novo modeling technology. Assessment of recent rhodopsin-based homology modeling studies indicates that, despite significant progress, these models do not yield hit rates that are sufficiently high for in silico screening (10 to 40% when screening for known binders). In contrast, the PREDICT modeling algorithm, which is independent of the rhodopsin structure, has now been fully validated in the context of drug discovery. PREDICT models are successfully used for drug discovery, yielding excellent hit rates (85 to 100% when screening for known binders), leading to the discovery of nanomolar-range new chemical entities for a variety of GPCR targets. Thus, 3D models of GPCRs should now allow the use of productive structure-based approaches for drug discovery.     

Yellow submarine of the Wnt/Frizzled signaling: submerging from the G protein harbor to the targets

The Wnt/Frizzled signaling pathway plays multiple functions in animal development and, when deregulated, in human disease. The G-protein coupled receptor (GPCR) Frizzled and its cognate heterotrimeric Gi/o proteins initiate the intracellular signaling cascades resulting in cell fate determination and polarization. In this review, we summarize the knowledge on the ligand recognition, biochemistry, modifications and interacting partners of the Frizzled proteins viewed as GPCRs. We also discuss the effectors of the heterotrimeric Go protein in Frizzled signaling. One group of these effectors is represented by small GTPases of the Rab family, which amplify the initial Wnt/Frizzled signal. Another effector is the negative regulator of Wnt signaling Axin, which becomes deactivated in response to Go action. The discovery of the GPCR properties of Frizzled receptors not only provides mechanistic understanding to their signaling pathways, but also paves new avenues for the drug discovery efforts.