G proteins in drug screening: from analysis of receptor-G protein specificity to manipulation of GPCR-mediated signalling pathways

Seven transmembrane G protein coupled receptors (7TM GPCRs) represent one of the largest gene familes in the human genome. Because of the size of the GPCR family, their proven history of being valuable targets for small molecule drug design, the fact that the absolute number of GPCRs that are targets for current medicines represents only a small fraction of the total encoded by the human genome, and that ligands for GPCRs do not have to enter the cell to exert their function, it is very likely that GPCRs will remain major targets for the pharmaceutical industry in the foreseeable future. Despite recent evidence indicating that GPCRs can provide information to cells, that does not require activation of G proteins ("signaling at zero G"), most of the GPCRs known to date function via interaction with and activation of heterotrimeric (alphabetagamma) G proteins. Thus, assay systems translating ligand modulation of GPCRs into G protein-dependent intracellular responses are a key component of both basic research and the drug discovery process. This article will review the current knowledge and recent progress in understanding molecular aspects of specific receptor-G protein recognition. It will also highlight how the knowledge generated by such studies can be transformed into assay systems for GPCR drug discovery.     

The significance of G protein-coupled receptor crystallography for drug discovery

Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.     

GPCR-Galpha fusion proteins: molecular analysis of receptor-G-protein coupling.

The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR-Galpha fusion proteins, which are formed by the fusion of GPCR to Galpha. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Galpha and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Galpha enhances coupling efficiency can be used to study aspects of receptor-G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR-Galpha fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.     

X-ray structure breakthroughs in the GPCR transmembrane region

G-protein-coupled receptor (GPCR) proteins [Lundstrom KH, Chiu ML, editors. G protein-coupled receptors in drug discovery. CRC Press; 2006] are the single largest drug target, representing 25-50% of marketed drugs [Overington JP, Al-Lazikani B, Hopkins AL. How many drug targets are there? Nat Rev Drug Discov 2006;5(12):993-6; Parrill AL. Crystal structures of a second G protein-coupled receptor: triumphs and implications. ChemMedChem 2008;3:1021-3]. While there are six subclasses of GPCR proteins, the hallmark of all GPCR proteins is the transmembrane-spanning region. The general architecture of this transmembrane (TM) region has been known for some time to contain seven α-helices. From a drug discovery and design perspective, structural information of the GPCRs has been sought as a tool for structure-based drug design. The advances in the past decade of technologies for structure-based design have proven to be useful in a number of areas. Invoking these approaches for GPCR targets has remained challenging. Until recently, the most closely related structures available for GPCR modeling have been those of bovine rhodopsin. While a representative of class A GPCRs, bovine rhodopsin is not a ligand-activated GPCR and is fairly distant in sequence homology to other class A GPCRs. Thus, there is a variable degree of uncertainty in the use of the rhodopsin X-ray structure as a template for homology modeling of other GPCR targets. Recent publications of X-ray structures of class A GPCRs now offer the opportunity to better understand the molecular mechanism of action at the atomic level, to deploy X-ray structures directly for their use in structure-based design, and to provide more promising templates for many other ligand-mediated GPCRs. We summarize herein some of the recent findings in this area and provide an initial perspective of the emerging opportunities, possible limitations, and remaining questions. Other aspects of the recent X-ray structures are described by Weis and Kobilka [Weis WI, Kobilka BK. Structural insights into G-protein-coupled receptor activation. Curr Opin Struct Biol 2008;18:734-40] and Mustafi and Palczewski [Mustafi D, Palczewski K. Topology of class A G protein-coupled receptors: insights gained from crystal structures of rhodopsins, adrenergic and adenosine receptors. Mol Pharmacol 2009;75:1-12].     

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.     

G protein-coupled receptors: novel targets for drug discovery in cancer

G protein-coupled receptors (GPCRs) belong to a superfamily of cell surface signalling proteins that have a pivotal role in many physiological functions and in multiple diseases, including the development of cancer and cancer metastasis. Current drugs that target GPCRs - many of which have excellent therapeutic benefits - are directed towards only a few GPCR members. Therefore, huge efforts are currently underway to develop new GPCR-based drugs, particularly for cancer. We review recent findings that present unexpected opportunities to interfere with major tumorigenic signals by manipulating GPCR-mediated pathways. We also discuss current data regarding novel GPCR targets that may provide promising opportunities for drug discovery in cancer prevention and treatment.     

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.     

Heterodimerisation of G protein-coupled receptors: implications for drug design and ligand screening

Importance of the field: In recent times many G protein-coupled receptors (GPCRs) have been shown to dimerise/oligomerise and, in some cases, such structural organization has been found to be essential for receptor function or to play a modulatory role in living cells. The fact that these complexes may display differential pharmacology through, for example, the formation of a new binding pocket or signalling properties, as well as different functions or regulation in physiological tissues, offers novel opportunities for drug discovery. As a consequence, it seems necessary to develop new approaches suitable for GPCR heterodimer identification and selective ligand screening. Areas covered in this review: This review gives an overview of new strategies that have been developed in an effort to incorporate the possibilities added by GPCR hetero-oligomerisation on the screening of compounds as drug candidates. What the reader will gain: The reader will gain a wider knowledge about how the current understanding of GPCR oligomeric structure and function has mandated that hetero-oligomeric receptors must be considered as novel targets in the identification of future lead compounds. Take home message: For the improvement of novel drug discovery, more structural and functional information on the process of receptor oligomerisation is needed, and the realisation that the function of GPCRs can be greatly influenced by other interacting receptors or proteins also demands consideration in the lead-compound developing process in order to achieve better therapeutic agents.     

Tools for GPCR drug discovery

G-protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic targets for a broad spectrum of diseases. The design and implementation of high-throughput GPCR assays that allow the cost-effective screening of large compound libraries to identify novel drug candidates are critical in early drug discovery. Early functional GPCR assays depend primarily on the measurement of G-protein-mediated 2nd messenger generation. Taking advantage of the continuously deepening understanding of GPCR signal transduction, many G-protein-independent pathways are utilized to detect the activity of GPCRs, and may provide additional information on functional selectivity of candidate compounds. With the combination of automated imaging systems and label-free detection systems, such assays are now suitable for high-throughput screening (HTS). In this review, we summarize the most widely used GPCR assays and recent advances in HTS technologies for GPCR drug discovery.     

Modulating G-protein-coupled receptors: from traditional pharmacology to allosterics

Signal transduction is the means by which cells respond to variations in their environment. G-protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors, accounting for >1% of the human genome. GPCRs respond to a wide variety of extracellular signals, including peptides, ions, amino acids, hormones, growth factors, light and odorant molecules. The receptors couple with heterotrimeric G proteins to transduce their signal across the membrane and into the cell. This coupling promotes the exchange of GDP for GTP on the Galpha subunit, leading to effector activation by both Galpha-GTP and Gbetagamma. Functional selectivity, whereby conformational changes in GPCRs induced by agonist binding lead to unique conformations that can differentially modulate the G protein coupling process, was first proposed over a decade ago. The implications are far reaching in pharmacology, as it means that a GPCR could have a different pharmacological profile depending on which G protein is activated and that the same GPCR could have different roles depending on the activating molecule as well as the G proteins present in the local environment.     

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.     

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.     

Ggamma-like (GGL) domains: new frontiers in G-protein signaling and beta-propeller scaffolding

The standard model of signal transduction from G-protein-coupled receptors (GPCRs) involves guanine nucleotide cycling by a heterotrimeric G-protein assembly composed of Galpha, Gbeta, and Ggamma subunits. The WD-repeat beta-propeller protein Gbeta and the alpha-helical, isoprenylated polypeptide Ggamma are considered obligate dimerization partners; moreover, conventional Gbetagamma heterodimers are considered essential to the functional coupling of Galpha subunits to receptors. However, our recent discovery of a Gbeta5 binding site (the Ggamma-like or "GGL" domain) within several regulators of G-protein signaling (RGS) proteins revealed the potential for functional GPCR/Galpha coupling in the absence of a conventional Ggamma subunit. In addition, we posit that the interaction between Gbeta5 isoforms and the GGL domains of RGS proteins represents a general mode of binding between beta-propeller proteins and their partners, extending beyond the realm of G-protein-linked signal transduction.     

Characterization of CHO cells stably expressing a G alpha 16/z chimera for high throughput screening of GPCRs

G protein-coupled receptors (GPCRs) are important therapeutic targets for drug discovery. The identification and characterization of new ligands ideally requires the use of high throughput assays that are applicable to all GPCR subtypes. To circumvent the problem of different GPCRs coupling to distinct intracellular second messenger pathways, we describe a new method that uses the chimeric Galpha protein 16z25 to facilitate this process. Stably expressed in Chinese hamster ovary cells, 16z25 allows G(i/o)- and G(s)-coupled receptors to mobilize intracellular Ca(2+) upon agonist stimulation. We have generated nine cell lines each stably expressing 16z25 and a GPCR. All cell lines respond to appropriate agonist stimulation in fluorometric imaging plate reader (FLIPR) assays with robust and potent Ca(2+) mobilization. Several of these lines have been pharmacologically characterized using agonists and antagonists. We also demonstrate that the coexpression of GPCR and 16z25 does not interfere with the receptors' ability to activate endogenous signaling pathways. The ability of 16z25 to functionally mediate the agonist stimulation of a broad spectrum of GPCRs indicates that the use of cell lines stably coexpressing this chimera and GPCRs will simplify the drug screening process and aid in the deorphanization of new receptors.     

针对G蛋白偶联受体的药物筛选新方法

G蛋白偶联受体（GPCR）为具有7个跨膜螺旋的蛋白质受体，是人体内最大的蛋白质家族，其为极重要的药物靶点。本文针对GPCR的固有激活和变构效应的药物筛选模型开发新进展和高内涵药物筛选新技术进行综述。     

Regulators of G-protein signaling and their Gα substrates: promises and challenges in their use as drug discovery targets

Because G-protein coupled receptors (GPCRs) continue to represent excellent targets for the discovery and development of small-molecule therapeutics, it is posited that additional protein components of the signal transduction pathways emanating from activated GPCRs themselves are attractive as drug discovery targets. This review considers the drug discovery potential of two such components: members of the "regulators of G-protein signaling" (RGS protein) superfamily, as well as their substrates, the heterotrimeric G-protein α subunits. Highlighted are recent advances, stemming from mouse knockout studies and the use of "RGS-insensitivity" and fast-hydrolysis mutations to Gα, in our understanding of how RGS proteins selectively act in (patho)physiologic conditions controlled by GPCR signaling and how they act on the nucleotide cycling of heterotrimeric G-proteins in shaping the kinetics and sensitivity of GPCR signaling. Progress is documented regarding recent activities along the path to devising screening assays and chemical probes for the RGS protein target, not only in pursuits of inhibitors of RGS domain-mediated acceleration of Gα GTP hydrolysis but also to embrace the potential of finding allosteric activators of this RGS protein action. The review concludes in considering the Gα subunit itself as a drug target, as brought to focus by recent reports of activating mutations to GNAQ and GNA11 in ocular (uveal) melanoma. We consider the likelihood of several strategies for antagonizing the function of these oncogene alleles and their gene products, including the use of RGS proteins with Gα(q) selectivity.     

Developing novel antifungals: lessons from G protein-coupled receptors

Up to 1.5 million people die yearly from fungal disease, but the repertoire of antifungal drug classes is minimal and the incidence of drug resistance is rising rapidly. This dilemma was recently declared by the World Health Organization as a global health emergency, but the discovery of new antifungal drug classes remains excruciatingly slow. This process could be accelerated by focusing on novel targets, such as G protein-coupled receptor (GPCR)-like proteins, that have a high likelihood of being druggable and have well-defined biology and roles in disease. We discuss recent successes in understanding the biology of virulence and in structure determination of yeast GPCRs, and highlight new approaches that might pay significant dividends in the urgent search for novel antifungal drugs.     

Signal transduction pathways of G protein-coupled receptors and their cross-talk with receptor tyrosine kinases: lessons from bradykinin signaling

G protein-coupled receptors (GPCRs) represent a major class of drug targets. Recent investigation of GPCR signaling has revealed interesting novel features of their signal transduction pathways which may be of great relevance to drug application and the development of novel drugs. Firstly, a single class of GPCRs such as the bradykinin type 2 receptor (B2R) may couple to different classes of G proteins in a cell-specific and time-dependent manner, resulting in simultaneous or consecutive initiation of different signaling chains. Secondly, the different signaling pathways emanating from one or several GPCRs exhibit extensive cross-talk, resulting in positive or negative signal modulation. Thirdly, GPCRs including B2R have the capacity for generation of mitogenic signals. GPCR-induced mitogenic signaling involves activation of the p44/p42 "mitogen activated protein kinases" (MAPK) and frequently "transactivation" of receptor tyrosine kinases (RTKs), an unrelated class of receptors for mitogenic polypeptides, via currently only partly understood pathways. Cytoplasmic tyrosine kinases and protein-tyrosine phosphatases (PTPs) which regulate RTK signaling are likely mediators of RTK transactivation in response to GPCRs. Finally, GPCR signaling is the subject of regulation by RTKs and other tyrosine kinases, including tyrosine phosphorylation of GPCRs itself, of G proteins, and of downstream molecules such as members of the protein kinase C family. In conclusion, known agonists of GPCRs are likely to have unexpected effects on RTK pathways and activators of signal-mediating enzymes previously thought to be exclusively linked to RTK activity such as tyrosine kinases or PTPs may be of much interest for modulating GPCR-mediated biological responses.     

Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling

G protein-coupled receptors (GPCRs) are seven transmembrane proteins that form the largest single family of integral membrane receptors. GPCRs transduce information provided by extracellular stimuli into intracellular second messengers via their coupling to heterotrimeric G proteins and the subsequent regulation of a diverse variety of effector systems. Agonist activation of GPCRs also initiates processes that are involved in the feedback desensitization of GPCR responsiveness, the internalization of GPCRs, and the coupling of GPCRs to heterotrimeric G protein-independent signal transduction pathways. GPCR desensitization occurs as a consequence of G protein uncoupling in response to phosphorylation by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes the binding of beta-arrestins, which not only uncouple receptors from heterotrimeric G proteins but also target many GPCRs for internalization in clathrin-coated vesicles. beta-Arrestin-dependent endocytosis of GPCRs involves the direct interaction of the carboxyl-terminal tail domain of beta-arrestins with both beta-adaptin and clathrin. The focus of this review is the current and evolving understanding of the contribution of GRKs, beta-arrestins, and endocytosis to GPCR-specific patterns of desensitization and resensitization. In addition to their role as GPCR-specific endocytic adaptor proteins, beta-arrestins also serve as molecular scaffolds that foster the formation of alternative, heterotrimeric G protein-independent signal transduction complexes. Similar to what is observed for GPCR desensitization and resensitization, beta-arrestin-dependent GPCR internalization is involved in the intracellular compartmentalization of these protein complexes.     

Spatiotemporal GPCR signaling illuminated by genetically encoded fluorescent biosensors

G protein-coupled receptors (GPCRs) are ligand-activated cell membrane proteins and represent the most important class of drug targets. GPCRs adopt several active conformations that stimulate different intracellular G proteins (and other transducers) and thereby modulate second messenger levels, eventually resulting in receptor-specific cell responses. It is increasingly accepted that not only the type of active signaling protein but also the duration of its stimulation and the subcellular location from where receptors signal distinctly contribute to the overall cell response. However, the molecular principles governing such spatiotemporal GPCR signaling and their role in disease are incompletely understood. Genetically encoded, fluorescent biosensors—in particular for the GPCR/cAMP signaling axis—have been pivotal to the discovery and molecular understanding of novel concepts in spatiotemporal GPCR signaling. These include GPCR priming, location bias, and receptor-associated independent cAMP nanodomains. Here, we review such technologies that we believe will illuminate the spatiotemporal organization of other GPCR signaling pathways that define the complex signaling architecture of the cell.