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.     

Screening for GPCR Ligands Using Surface Plasmon Resonance

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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.     

Nonradioactive GTP binding assay to monitor activation of g protein-coupled receptors

GPCRs represent important targets for drug discovery because GPCRs participate in a wide range of cellular signaling pathways that play a role in a variety of pathological conditions. A large number of screening assays have been developed in HTS laboratories for the identification of hits or lead compounds acting on GPCRs. One type of assay that has found relatively widespread application, due to its at least in part generic nature, relies on the use of a radioactive GTP analogue, [(35)S]GTPgammaS. The G-protein alpha subunit is an essential part of the interaction between receptor and G proteins in transmembrane signaling, where the activated receptor catalyzes the release of GDP from Galpha, thereby enabling the subsequent binding of GTP or a GTP analogue. [(35)S]GTPgammaS allows the extent of this interaction to be followed quantitatively by determining the amount of radioactivity associated with cell membranes. However, with the increased desire to move assays to nonradioactive formats, there is a considerable need to develop a nonradioactive GTP binding assay to monitor ligand-induced changes in GPCR activity. The Eu-GTP binding assay described here is based on TRF that exploits the unique fluorescence properties of lanthanide chelates, and provides a powerful alternative to assays using radioisotopes. In this article, we have used the human alpha(2A)-AR as a model GPCR system to evaluate the usefulness of this Eu-GTP binding assay.     

Fragment-based lead discovery on G-protein-coupled receptors

Introduction: G-protein-coupled receptors (GPCRs) form one of the largest groups of potential targets for novel medications. Low druggability of many GPCR targets and inefficient sampling of chemical space in high-throughput screening expertise however often hinder discovery of drug discovery leads for GPCRs. Fragment-based drug discovery is an alternative approach to the conventional strategy and has proven its efficiency on several enzyme targets. Based on developments in biophysical screening techniques, receptor stabilization and in vitro assays, virtual and experimental fragment screening and fragment-based lead discovery recently became applicable for GPCR targets.

Areas covered: This article provides a review of the biophysical as well as biological detection techniques suitable to study GPCRs together with their applications to screen fragment libraries and identify fragment-size ligands of cell surface receptors. The article presents several recent examples including both virtual and experimental protocols for fragment hit discovery and early hit to lead progress.

Expert opinion: With the recent progress in biophysical detection techniques, the advantages of fragment-based drug discovery could be exploited for GPCR targets. Structural information on GPCRs will be more abundantly available for early stages of drug discovery projects, providing information on the binding process and efficiently supporting the progression of fragment hit to lead. In silico approaches in combination with biological assays can be used to address structurally challenging GPCRs and confirm biological relevance of interaction early in the drug discovery project.     

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.     

A fully automated [35S]GTPgammaS scintillation proximity assay for the high-throughput screening of Gi-linked G protein-coupled receptors

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.     

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.     

Strategies for the identification of allosteric modulators of G-protein-coupled receptors

Once considered a pharmacological curiosity, allosteric modulation of seven transmembrane domain G-protein-coupled receptors (GPCRs) has emerged as a potentially powerful means to affect receptor function for therapeutic purposes. Allosteric modulators, which interact with binding sites topologically distinct from the orthosteric ligand binding sites, can potentially provide improved selectivity and safety, along with maintenance of spatial and temporal aspects of GPCR signaling. Accordingly, drug discovery efforts for GPCRs have increasingly focused on the identification of allosteric modulators. This review is devoted to an examination of the strategies, challenges, and opportunities for high-throughput screening for allosteric modulators of GPCRs, with particular focus on the identification of positive allosteric modulators.     

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.     

Receptor-ligand docking simulation for membrane proteins

Protein structure-based molecular design using the computational techniques of protein structure prediction, ligand docking, and virtual screening is an integral part of drug discovery for limiting the application of the structure-based approach to target proteins such as G-protein-coupled receptors (GPCRs). GPCRs play an important role in living organisms and are of major interest to the pharmaceutical industry. However, structural data on ligand-binding forms for GPCRs from experiments to elucidate structural templates for docking simulations are lacking due to the difficulties associated with crystallization and crystallography. Therefore structural prediction of GPCRs in the ligand-bound state using computational methods has been introduced, but the prediction of ligand conformation onto target GPCRs is still constructed manually by human experts. We developed a molecular modeling technique for the prediction of ligand-receptor binding using comparative ligand-binding analysis (CoLBA) that not only considers interaction energy but also the similarity of interaction profiles among ligands. The advantage of CoLBA is that it can facilitate intuitive and flexible screening based on docking results when protein structures with low resolution (or theoretical models) are targeted. We applied CoLBA to ligand-binding prediction in several GPCRs. The predicted ligand-binding models were evaluated in site-directed mutagenesis experiments in collaborative research, and the enrichment rate of activated ligands was compared with random compounds in virtual screening simulations. We propose that CoLBA can be applied in large-scale modeling of ligand-receptor complexes and virtual screening for GPCRs.     

G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey

G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor.     

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.     

G-protein-coupled receptor heterodimerization: assay technologies to clinical significance

The concept of G-protein-coupled receptor (GPCR) oligomerization has existed for many years, but has only recently received scrutiny from the pharmaceutical industry. The new interest in this theory has arisen not only because technologies to exploit GPCR oligomers are now available, but also because GPCR oligomerization has the potential to explain previously anomalous pharmacology and to provide an array of new therapeutic targets. The emergence of higher order GPCR organization adds a layer of complexity in the context of cellular control, and indeed drug discovery. This review outlines the current knowledge surrounding the oligomerization of GPCRs and describes effective assays for exploiting oligomerization in drug discovery. The clinical significance of oligomerization and the most promising strategies for therapeutically targeting GPCR oligomers are discussed.     

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 use of constitutively active receptors for drug discovery at the G protein-coupled receptor gene pool

Several lines of evidence over the last decade have established that G protein-coupled receptors (GPCRs) can signal in the absence of their natural ligand which results in ligand-independent or constitutive activity. Natural genetic mutation, overexpression and site-directed mutagenesis all result in constitutive activation of GPCRs. Of the 100 leading pharmaceutical products in 2000, 39, wholly or in part, acted through a GPCR-mediated mechanism, a fact that underlines the extreme importance of GPCRs as pharmaceutical drug targets. In addition, the sequencing of the human genome and database mining has revealed that there are hundreds of putative orphan GPCRs for which the natural ligands have not been identified. These orphan GPCRs have largely been inaccessible to drug discovery because traditional methods have mainly relied on ligand-dependent binding assays to discover and pharmacologically characterize potential drug candidates from this receptor class. In the absence of ligand identification, constitutively active receptors allow for a logical and direct way forward through the drug discovery pathway by providing the tool necessary to find modulators of this receptor class in a ligand-independent fashion.     

Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery

Fluorescence methods are commonly used in pharmaceutical drug discovery to assay the binding of drug-like compounds to signaling proteins and other bio-particles. For binding studies of non-fluorescent compounds, a competitive format may be used in which the binding of the compound results in displacement of another fluorescently labeled ligand. Highly-sensitive measurements within nano-liter sized open probe volumes can be accomplished using a confocal epi-illumination geometry and thus key tools for such drug-binding studies include fluorescence correlation spectroscopy (FCS) and its related techniques. This paper reviews the general protocol for application of FCS to biomolecular compound-binding assays and it focuses on methods for the reduction of experimental photon count data to obtain the normalized autocorrelation function (ACF), on theoretical models of the ACF, and on statistical and systematic errors in the experimental ACF. Results from a detailed Monte Carlo simulation of FCS, which are useful for testing theoretical models and validating short-duration assay capabilities, are discussed. An illustrative example is presented on the use of FCS to assay binding of Alexa-488-labeled Bak peptide with Bcl-x(L), which is an intracellular protein that acts to protect against programmed cell death.     

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.     

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.     

Discovery of β2 Adrenergic Receptor Ligands Using Biosensor Fragment Screening of Tagged Wild-Type Receptor

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