Identification of endogenous surrogate ligands for human P2Y receptors through an in silico search

G protein-coupled receptors (GPCRs) are distributed widely throughout the human body, and nearly 50% of current medicines act on a GPCR. GPCRs are considered to consist of seven transmembrane alpha-helices that form an alpha-helical bundle in which agonists and antagonists bind. A 3D structure of the target GPCR is indispensable for designing novel medicines acting on a GPCR. We have previously constructed the 3D structure of human P2Y(1) (hP2Y(1)) receptor, a GPCR, by homology modeling with the 3D structure of bovine rhodopsin as a template. In the present study, we have employed an in silico screening for compounds that could bind to the hP2Y(1)-receptor model using AutoDock 3.0. We selected 21 of the 30 top-ranked compounds, and by measuring intracellular Ca(2+) concentration, we identified 12 compounds that activated or blocked the hP2Y(1) receptor stably expressed in recombinant CHO cells. 5-Phosphoribosyl-1-pyrophosphate (PRPP) was found to activate the hP2Y(1) receptor with a low ED(50) value of 15 nM. The Ca(2+) assays showed it had no significant effect on P2Y(2), P2Y(6), or P2X(2) receptors, but acted as a weak agonist on the P2Y(12) receptor. This is the first study to rationally identify surrogate ligands for the P2Y-receptor family.     

G蛋白偶联受体固有活性研究进展与新药开发

G蛋白偶联受体(G-prote in-coup led receptor,GPCR)是与G蛋白有信号连接的一大类受体家族,是人体内最大的膜受体蛋白家族,是一类具有7个跨膜螺旋的跨膜蛋白受体。GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标。目前世界药物市场上有三分之一的小分子药物是GPCR的激活剂(agon ist)或拮抗剂(antagon ist)。以其为靶点的药物在医药产业中占据显著地位。在当今前50种最畅销的上市药物中,20%属于G蛋白受体相关药物。近来的研究发现,大多数G蛋白偶联受体具有一个很重要的特性,就是具有固有活性(Constitutive ac-tivity),即无激动剂条件受体自发的维持激活并维持下游信号传导通路的活性。固有活性涉及受体、G蛋白及下游信号通路之间的关系。该文就G蛋白偶联受体固有活性概念、研究进展、反相激动剂与固有活性研究、固有活性与新药开发4个方面,进行以下论述。     

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.     

Ligand-directed trafficking of receptor stimulus

GPCRs are seven transmembrane-spanning receptors that convey specific extracellular stimuli to intracellular signalling. They represent the largest family of cell surface proteins that are therapeutically targeted. According to the traditional two-state model of receptor theory, GPCRs were considered as operating in equilibrium between two functional conformations, an active (R*) and inactive (R) state. Thus, it was assumed that a GPCR can exist either in an “off” or “on” conformation causing either no activation or equal activation of all its signalling pathways. Over the past several years it has become evident that this model is too simple and that GPCR signalling is far more complex. Different studies have presented a multistate model of receptor activation in which ligand-specific receptor conformations are able to differentiate between distinct signalling partners. Recent data show that beside G proteins numerous other proteins, such as β-arrestins and kinases, may interact with GPCRs and activate intracellular signalling pathways. GPCR activation may therefore involve receptor desensitization, coupling to multiple G proteins, Gα or Gβγ signalling, and pathway activation that is independent of G proteins. This latter effect leads to agonist “functional selectivity” (also called ligand-directed receptor trafficking, stimulus trafficking, biased agonism, biased signalling), and agonist intervention with functional selectivity may improve the therapy. Many commercially available drugs with beneficial efficacy also show various undesirable side effects. Further studies of biased signalling might facilitate our understanding of the side effects of current drugs and take us to new avenues to efficiently design pathway-specific medications.     

CypHer 5: a generic approach for measuring the activation and trafficking of G protein-coupled receptors in live cells

GPCRs are one of the most popular classes of therapeutic drug targets. It is therefore important to design specific assay formats to readily identify ligands at these receptors. CypHer 5 technology utilizes the general ability of GPCRs to be internalized into the endosomal pathway of a cell in response to agonist ligands. The CypHer 5 dye is fluorescent in acidic environments, but nonfluorescent at neutral pH. When CypHer 5 is bound to a receptor on the extracellular surface of the cell, it is essentially nonfluorescent. On internalization into a cell, it displays a significant increase in fluorescence. Here we demonstrate the detection of agonist activation of two GPCRs in stably transfected live cells using CypHer 5 technology. The G(q)-coupled TRHR-1 and the G(s)-coupled beta(2)-adrenoceptor were both N-terminally tagged with VSV-G. Following addition of CypHer 5-labeled anti-VSV-G antibodies to HEK 293 cells stably expressing the beta(2)-adrenoceptor or CHO-K1 cells stably expressing the TRHR-1, the cells were treated with agonists and then imaged on Amersham Biosciences' IN Cell Analyzer 3000. Data were quantified using a granularity analysis module. Concentration-response curves were obtained with signal-to-background ratios of 7:1 for both receptors. An EC(50) of 0.52 nM was observed on TRH stimulation of the TRHR-1, and an EC(50) of 30 nM was obtained on isoprenaline stimulation of the beta(2)-adrenoceptor. These results demonstrated that the CypHer technology was capable of measuring high-potency agonist responses. The beta(2)-adrenoceptor antagonist, alprenolol, competed for isoprenaline with an IC(50) of 30 nM, indicating that a high-potency antagonist inhibition curve could also be observed using CypHer. CypHer 5 provides a generic tool to measure GPCR activation in a live cell, homogeneous assay format, and may be equally suitable for detecting activation of other classes of cell surface receptors.     

A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation

Guanosine triphosphate binding protein (G protein)-coupled receptors (GPCRs) are a large class of pharmaceutical drug targets. With the increasing popularity of functional assays for high throughput screening, there arises an increasing need for robust second messenger assays that reflect GPCR activation and are readily amenable for miniaturization. GPCRs that upon agonist stimulation modulate adenylyl cyclase activity, and, consequently, cellular cyclic adenosine monophosphate (cAMP) levels, via the G protein Gs or Gi, form a subset of therapeutic targets. While there are several cAMP assays currently available, most are not scalable for miniaturization into the 1536-well format employed for automated high throughput screening of large chemical libraries. Here, we describe a cAMP assay based on the enzyme fragmentation complementation (EFC) of beta-galactosidase. In this assay, recombinant cells expressing Gs- or Gi-coupled receptors exhibit robust and reproducible pharmacology for agonists and antagonists, as measured by cAMP levels. Furthermore, the EFC cAMP assay offers sufficient sensitivity to be used with cells expressing endogenous GPCRs. We demonstrate the miniaturization of this assay into a 1536-well format with comparable sensitivity and plate statistics to those of the 384-well assay for both Gs- and Gi-coupled receptors, and its suitability for miniaturized high throughput screening.     

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.     

New insights from structural biology into the druggability of G protein-coupled receptors

The recent availability of X-ray structures for diverse ligand-bound Family A G protein-coupled receptors (GPCRs) in multiple conformations (inactive form with an antagonist/inverse agonist bound and active form with an agonist bound) now enables rational drug design efforts that have historically been applied to soluble enzyme targets. Here, we review properties of these GPCR binding sites, using a unique combination of calculated physicochemical properties and water energetics (GRID, WaterMap and SZMAP) to provide a new perspective and rational assessment of druggability for each GPCR target binding site. Examples are described from several well-studied enzyme systems to support this advanced structure-based approach to assessing druggability and to contrast their properties with those of GPCRs. Changes in receptor conformations between the GPCR inactive and active forms evident from the protein structures are discussed, yielding important pointers for rational drug design of antagonists and agonists and a better understanding of GPCR activation.     

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.     

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.     

An update of novel screening methods for GPCR in drug discovery

Introduction: G protein-coupled receptors (GPCRs) are the largest and most versatile group of cytomembrane receptors, comprising of approximately 300 non-sensory and druggable members. Traditional GPCR drug screening is based on radiometric competition binding assays, which are expensive and hazardous to human health. Furthermore, the paradox of high investment and low output, in terms of new drugs, highlights the need for more efficient and effective drug screening methods. Areas covered: This review summarizes non-radioactive assays assessing the ligand-receptor binding including: the fluorescence polarization assay, the TR-FRET assay and the surface plasmon resonance assay. It also looks at non-radioactive assays that assess receptor activation and signaling including: second messenger-based assays and β-arrestin recruitment-based assays. This review also looks at assays based on cellular phenotypic change. Expert opinion: GPCR signaling pathways look to be more complicated than previously thought. The existence of receptor allosteric sites and multireceptor downstream effectors restricts the traditional assay methods. The emergence of novel drug screening methods such as those for assessing β-arrestin recruitment and cellular phenotypic change may provide us with improved drug screening efficiency and effect.     

Novel roles for arrestins in G protein-coupled receptor biology and drug discovery

The G protein-coupled receptors (GPCRs) are the largest family of membrane proteins and represent some of the most important pharmaceutical targets. These receptors, encoded by several hundred genes, are activated by a wide variety of endogenous and synthetic ligands. The study of the signal transduction pathways activated by these receptors and the associated mechanisms controlling biological responses have been pivotal in identifying key intracellular molecules for regulating receptor responsiveness. The beta-arrestin proteins, which were initially discovered due to their role in GPCR desensitization, serve equally important roles in regulating internalization and alternative signaling events. This review focuses on the different functions of beta-arrestins to demonstrate how these proteins can help to identify new ligands for GPCRs and how they can serve as a platform for drug discovery.     

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.     

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.     

Crosstalk within GPCR heteromers in schizophrenia and Parkinson's disease: physical or just functional?

Crosstalk between G protein-coupled receptors (GPCRs) is one of the key mechanisms used by the cell for integrating multiple signaling pathways. Functional crosstalk at the level of signaling pathways was initially thought to regulate receptor function. Importantly, the existence of GPCR heteromers demonstrates that direct physical interactions between GPCRs could also be behind the crosstalk phenomenon. Neurological disorders such as Parkinson's disease (PD) and schizophrenia have been linked to a dysfunctional communication between certain GPCRs. In this review, we discuss functional and physical crosstalk of the main GPCR families involved in the aforementioned disorders. In addition, we analyze the available structural information on physical crosstalk and highlight some strategies in drug discovery based on these crosstalk mechanisms.     

Allosteric modulators of G-protein-coupled receptors

Allosteric modulators of G-protein-coupled receptors (GPCRs) interact with binding sites on the receptor that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Allosteric modulators offer several advantages over standard orthosteric drugs, including the potential for greater receptor subtype selectivity. To date, the current paucity of clinically available allosteric drugs reflects the bias of traditional radioligand binding assays towards the detection of orthosteric effects. However, the advent of new cell-based high-throughput functional assays has led to an increased detection of allosteric GPCR ligands. The current challenge for modulator-based GPCR drug discovery is the optimization of both binding and functional assays to better detect and validate allosteric ligands.     

Allosteric modulation of heterodimeric G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) are, and will probably remain, the most tractable class of targets for the development of small-molecule therapeutic medicines. Currently, all approved GPCR-directed medicines are agonists or antagonists at orthosteric binding sites - except for the calcimimetic cinacalcet, which is a positive allosteric modulator of Ca(2+)-sensing receptors, and maraviroc, an allosteric inhibitor of CC-chemokine receptor (CCR) 5. It is now widely accepted that GPCRs exist and might function as dimers, and there is growing evidence for the physiological presence and relevance of GPCR heterodimers. Molecules that can regulate a GPCR within a heterodimer, through allosteric effects between the two protomers of the dimer or between a protomer or protomers and the associated G protein, offer the potential to function in a highly selective and tissue-specific way. Despite the conceptual attraction of such allosteric regulators of GPCR heterodimers as drugs, they cannot be identified by screening approaches that routinely use a 'one GPCR target at a time' strategy. In our opinion, this will require the development of new approaches for screening and a return to the use of physiologically relevant cell systems at an early stage in compound identification.     

Agonist-dependent modulation of G protein-coupled receptor kinase 2 by mitogen-activated protein kinases

A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed beta-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and beta-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of beta(2)-adrenergic receptors (beta(2)-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with beta(2)-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of beta(2)-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gbetagamma-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.     

Ubiquitination of g protein-coupled receptors: functional implications and drug discovery

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of signaling receptors and control a vast array of physiological responses. Modulating the signaling responses of GPCRs therapeutically is important for the treatment of various diseases, and discovering new aspects of GPCR signal regulation is critical for future drug development. Post-translational modifications are integral to the regulation of GPCR function. In addition to phosphorylation, many GPCRs are reversibly modified with ubiquitin. Ubiquitin is covalently attached to lysine residues within the cytoplasmic domains of GPCRs by ubiquitin ligases and removed by ubiquitin-specific proteases. In many cases, ubiquitin functions as a sorting signal that facilitates trafficking of mammalian GPCRs from endosomes to lysosomes for degradation, but not all GPCRs use this pathway. Moreover, there are distinct types of ubiquitin conjugations that are known to serve diverse functions in controlling a wide range of cellular processes, suggesting broad roles for GPCR ubiquitination. In this review, we highlight recent studies that illustrate various roles for ubiquitin in regulation of GPCR function. Ubiquitination is known to target many GPCRs for lysosomal degradation, and current studies now indicate that basal ubiquitination, deubiquitination, and transubiquitination of certain GPCRs are important for controlling cell surface expression and cellular responsiveness. In addition, novel functions for ubiquitin in regulation of GPCR dimers and in mediating differential GPCR regulation induced by biased agonists have been reported. We will discuss the implications of these new discoveries for ubiquitin regulation of GPCR function in the context of drug development.     

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.