Techniques: promiscuous Galpha proteins in basic research and drug discovery

Assay technologies that measure the activation of heterotrimeric (alphabetagamma) G proteins by G-protein-coupled receptors (GPCRs) are well established within the pharmaceutical industry, either for pharmacological characterization or for the identification of natural or surrogate receptor ligands. Despite recent evidence indicating that GPCR-linked signalling events might not be mediated exclusively by G proteins, G-protein activation remains a common benchmark for assessing GPCR family members. Thus, assay systems that translate ligand-mediated modulation of GPCRs into G-protein-dependent intracellular responses still represent key components of both basic research and the drug discovery process. In this article, the current knowledge and recent progress of integrating Galpha subunits into assay systems for GPCR drug discovery will be reviewed. Emphasis is given to novel promiscuous and chimeric Galpha proteins. Because of their ability to interact with a wide range of GPCRs, such novel G proteins are likely to be incorporated rapidly into drug discovery programmes.     

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。     

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.     

Computational identification and analysis of G protein‐coupled receptor targets

The completion of the human genome sequence has provided a large pool of potential drug targets for disease therapy. G protein–coupled receptors (GPCRs), which are central to signaling networks that regulate basic cellular processes, represent the most important known class of therapeutic targets for multiple disease states. Bioinformatics approaches can be applied to facilitate the identification of novel GPCRs, understanding their physiological and pathological roles, and screening for drug discovery. The present review summarizes current bioinformatics approaches that can be used to identify and analyze GPCR targets. In addition, the limitations of these technologies with the intention of setting reasonable expectations are also discussed together with some potential avenues for GPCR research. Drug Dev. Res. 67:771–780, 2006. © 2007 Wiley‐Liss, Inc.     

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.     

Structure-based drug screening for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) represent a large family of signaling proteins that includes many therapeutic targets; however, progress in identifying new small molecule drugs has been disappointing. The past 4 years have seen remarkable progress in the structural biology of GPCRs, raising the possibility of applying structure-based approaches to GPCR drug discovery efforts. Of the various structure-based approaches that have been applied to soluble protein targets, such as proteases and kinases, in silico docking is among the most ready applicable to GPCRs. Early studies suggest that GPCR binding pockets are well suited to docking, and docking screens have identified potent and novel compounds for these targets. This review will focus on the current state of in silico docking for GPCRs.     

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.     

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.     

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.     

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.     

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.     

G蛋白信号调节蛋白作为中枢神经系统药物新靶点研究进展

G蛋白偶联受体(G-prote in-coup led receptors,GPCR)是许多治疗药物的作用靶点。G蛋白信号调节蛋白(regu latorof G-prote in signaling,RGS)属一类新发现的蛋白家族,它们在GPCR信号传导中起重要作用。一般来说RGS可加速G蛋白失活进而终止GPCR信号传导,但也有些RGS同时具有效应分子和信号传递功能。兼具GPCR激动和RGS抑制功能的药物将大大增强信号传导,同时还能增加激动剂的区域特异性。由于RGS的多样性,组织分布特异性以及较强的调节活性,RGS很可能成为寻找新型中枢神经系统疾病治疗药物的新靶点。     

Emerging concepts of guanine nucleotide-binding protein-coupled receptor (GPCR) function and implications for high throughput screening

Guanine nucleotide binding protein (G protein) coupled receptors (GPCRs) comprise one of the largest families of proteins in the human genome and are a target for 40% of all approved drugs. GPCRs have unique structural motifs that allow them to interact with a wide and diverse series of extracellular ligands, as well as intracellular proteins, G proteins, receptor activity-modifying proteins, arrestins, and indeed other receptors. This distinctive structure has led to numerous efforts to discover drugs against GPCRs with targeted therapeutic uses. Such "designer" drugs currently include allosteric regulators, inverse agonists, and drugs targeting hetero-oligomeric complexes. Moreover, the large family of orphan GPCRs provides a rich and novel field of targets to discover drugs with unique therapeutic properties. The numerous technologies to discover GPCR drugs have also greatly advanced over the years, facilitating compound screening against known and orphan GPCRs, as well as in the identification of unique designer GPCR drugs. Indeed, high throughput screening (HTS) technologies employing functional cell-based approaches are now widely used. These include measurement of second messenger accumulation such as cyclic AMP, calcium ions, and inositol phosphates, as well as mitogen-activated protein kinase activation, protein-protein interactions, and GPCR oligomerization. This review focuses on how the improved understanding of the molecular pharmacology of GPCRs, coupled with a plethora of novel HTS technologies, is leading to the discovery and development of an entirely new generation of GPCR-based therapeutics.     

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.     

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].     

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.     

Structural diversity of G protein-coupled receptors and significance for drug discovery

G protein-coupled receptors (GPCRs) are the largest family of membrane-bound receptors and also the targets of many drugs. Understanding of the functional significance of the wide structural diversity of GPCRs has been aided considerably in recent years by the sequencing of the human genome and by structural studies, and has important implications for the future therapeutic potential of targeting this receptor family. This article aims to provide a comprehensive overview of the five main human GPCR families--Rhodopsin, Secretin, Adhesion, Glutamate and Frizzled/Taste2--with a focus on gene repertoire, general ligand preference, common and unique structural features, and the potential for future 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.     

Structure-based design in the GPCR target space

The G protein-coupled receptors (GPCRs) are membrane proteins that transmit signals via G-protein coupling. They have long been the target of small molecule therapeutics accounting for 30% of the launched drug targets. They are subdivided into several classes, with rhodopsins corresponding to the largest class. Furthermore, a high number of new rhodopsin-like GPCR proteins are included in the druggable genome, thus they are projected to continue being of value to the pharmaceutical and biotechnology sectors. We present a comprehensive review of the structural information pertaining to GPCRs, in light of the most recently deposited crystal structures, along with comparisons of the available to-date structures at different activation states. Finally, computational approaches to GPCR modeling are discussed in conjunction with critical perspectives regarding feasibility and limitations.     

Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance

After many years of effort, recent technical breakthroughs have enabled the X-ray crystal structures of three G-protein-coupled receptors (GPCRs) (β1 and β2 adrenergic and adenosine A_2a) to be solved in addition to rhodopsin. GPCRs, like other membrane proteins, have lagged behind soluble drug targets such as kinases and proteases in the number of structures available and the level of understanding of these targets and their interaction with drugs. The availability of increasing numbers of structures of GPCRs is set to greatly increase our understanding of some of the key issues in GPCR biology. In particular, what constitutes the different receptor conformations that are involved in signalling and the molecular changes which occur upon receptor activation. How future GPCR structures might alter our views on areas such as agonist-directed signalling and allosteric regulation as well as dimerization is discussed. Knowledge of crystal structures in complex with small molecules will enable techniques in drug discovery and design, which have previously only been applied to soluble targets, to now be used for GPCR targets. These methods include structure-based drug design, virtual screening and fragment screening. This review considers how these methods have been used to address problems in drug discovery for kinase and protease targets and therefore how such methods are likely to impact GPCR drug discovery in the future.This article is part of a themed section on Molecular Pharmacology of GPCR. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x