Label-free cell-based assays for GPCR screening

G protein-coupled receptors (GPCRs) have been proven to be the largest family of druggable targets in the human genome. Given the importance of GPCRs as drug targets and the de-orphanization of novel targets, GPCRs are likely to remain the frequent targets of many drug discovery programs. With recent advances in instrumentation and understanding of cellular mechanisms for the signals measured, biosensor-centered label-free cell assay technologies become a very active area for GPCR screening. This article reviews the principles and potential of current label-free cell assay technologies in GPCR drug discovery.     

High content screening for G protein-coupled receptors using cell-based protein translocation assays

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.     

Small Molecule Tools to Study Cellular Target Engagement for the Intracellular Allosteric Binding Site of GPCRs

A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein-coupled receptors (GPCRs). Ligands targeting the IABS, so-called intracellular allosteric antagonists, are highly promising compounds for pharmaceutical intervention and currently evaluated in several clinical trials. Beside co-crystal structures that laid the foundation for the structure-based development of intracellular allosteric GPCR antagonists, small molecule tools that enable an unambiguous identification and characterization of intracellular allosteric GPCR ligands are of utmost importance for drug discovery campaigns in this field. Herein, we discuss recent approaches that leverage cellular target engagement studies for the IABS and thus play a critical role in the evaluation of IABS-targeted ligands as potential therapeutic agents.     

GPCR–drug interactions prediction using random forest with drug-association-matrix-based post-processing procedure

G-protein-coupled receptors (GPCRs) are important targets of modern medicinal drugs. The accurate identification of interactions between GPCRs and drugs is of significant importance for both protein function annotations and drug discovery. In this paper, a new sequence-based predictor called TargetGDrug is designed and implemented for predicting GPCR–drug interactions. In TargetGDrug, the evolutionary feature of GPCR sequence and the wavelet-based molecular fingerprint feature of drug are integrated to form the combined feature of a GPCR–drug pair; then, the combined feature is fed to a trained random forest (RF) classifier to perform initial prediction; finally, a novel drug-association-matrix-based post-processing procedure is applied to reduce potential false positive or false negative of the initial prediction. Experimental results on benchmark datasets demonstrate the efficacy of the proposed method, and an improvement of 15% in the Matthews correlation coefficient (MCC) was observed over independent validation tests when compared with the most recently released sequence-based GPCR–drug interactions predictor. The implemented webserver, together with the datasets used in this study, is freely available for academic use at http://csbio.njust.edu.cn/bioinf/TargetGDrug.     

G Protein-coupled Receptor (GPCR) Reconstitution and Labeling for Solution Nuclear Magnetic Resonance (NMR) Studies of the Structural Basis of Transmembrane Signaling

G protein-coupled receptors (GPCRs) are a large membrane protein family found in higher organisms, including the human body. GPCRs mediate cellular responses to diverse extracellular stimuli and thus control key physiological functions, which makes them important targets for drug design. Signaling by GPCRs is related to the structure and dynamics of these proteins, which are modulated by extrinsic ligands as well as by intracellular binding partners such as G proteins and arrestins. Here, we review some basics of using nuclear magnetic resonance (NMR) spectroscopy in solution for the characterization of GPCR conformations and intermolecular interactions that relate to transmembrane signaling.     

Latest development in drug discovery on G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the family of proteins with the highest impact from social, therapeutic and economic point of view. Today, more than 50% of drug targets are based on GPCRs and the annual worldwide sales exceeds 50 billion dollars. GPCRs are involved in all major disease areas such as cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. The classical drug discovery process has relied on screening compounds, which interact favorably with the GPCR of interest followed by further chemical engineering as a mean of improving efficacy and selectivity. In this review, methods for sophisticated chemical library screening procedures will be presented. Furthermore, development of cell-based assays for functional coupling of GPCRs to G proteins will be discussed. Finally, the possibility of applying structure-based drug design will be summarized. This includes the application of bioinformatics knowledge and molecular modeling approaches in drug development programs. The major efforts established through large networks of structural genomics on GPCRs, where recombinantly expressed GPCRs are subjected to purification and crystallization attempts with the intention of obtaining high-resolution structures, are presented as a promising future approach for tailor-made drug development.     

Allosteric Optical Control of a Class B G‐Protein‐Coupled Receptor

Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G‐protein‐coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light‐activated positive allosteric modulator of the glucagon‐like peptide‐1 receptor (GLP‐1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca²⁺, cAMP, and insulin responses to glucagon‐like peptide‐1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small‐molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP‐1R.     

High throughput screening (HTS) in identification new ligands and drugable targets of G protein-coupled receptors (GPCRs)

G protein-coupled receptors (GPCRs) which constitute one of the largest and most versatile families of cell surface receptors are involved in a wide spectrum of physiological functions, such as, neuronal transmission, chemotaxis, pacemaker activity and embryonic development. Therefore, in the past a few years GPCR families have become very important targets in pharmaceutical design. However, according to the human genome project, there are approximately 1000 genes encoding GPCRs, only about 200 of GPCRs have known ligands and functions. Searching for ligands of the unknown GPCRs and better modulators of known GPCRs are currently attracting lots of interest. High throughput screening (HTS), which is commonly defined as an automatic process of testing potential drug candidates efficiently, is widely used in drug discovery. In this review, the use of high throughput screening (HTS) in studying GPCRs and the choice of screening technology in different G-protein signaling pathways were summarized.     

Using silico methods predicting ligands for orphan GPCRs

The G-protein coupled receptor (GPCR) superfamily is one of the most important drug target classes for the pharmaceutical industry. The completion of the human genome project has revealed that there are more than 300 potential GPCR targets of interest. The identification of their natural ligands can gain significant insights into regulatory mechanisms of cellular signaling networks and provide unprecedented opportunities for drug discovery. Much effort has been directed towards the GPCR ligand discovery study by both academic institutions and pharmaceutical industries. However, the endogenous ligands still remain unknown for about 150 GPCRs in the human genome. It is necessary to develop new strategies to predict candidate ligands for these so-called orphan receptors. Computational techniques are playing an increasingly important role in finding and validating novel ligands for orphan GPCRs (oGPCRs). In this paper, we focus on recent development in applying bioinformatics approaches for the discovery of GPCR ligands. In addition, some of the data resources for ligand identification are also provided.     

New strategies in drug discovery for GPCRs: high throughput detection of cellular ERK phosphorylation

G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Due to their broad involvement in cellular responses, GPCRs affect many important body functions both in health and disease. Compared to other receptor families, the GPCRs have been a rich source of extracellularly-acting pharmaceuticals, due largely to the fact that many GPCR ligands are small molecules when compared with ligands for other receptors, such as the tyrosine kinase receptor family. This has allowed the development of small molecule modulators of receptor function that act on specific GPCRs, such as those involved in cardiovascular regulation. However, at several levels, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs, such as the Gi-coupled GPCRs, are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca(2+) and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs is likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high throughput methodologies. This article presents recent advances and associated applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery.     

Membrane interactive alpha-helices in GPCRs as a novel drug target

G-Protein Coupled Receptors (GPCRs) are one of the most important targets for pharmaceutical drug design. Over the past 30 years, mounting evidence has suggested the existence of homo and hetero dimers or higher-order complexes (oligomers) that are involved in signal transduction and some diseases. The number of reports describing GPCR oligomerization has increased, and in 2003, the organization of mouse rhodopsin into two-dimensional arrays of dimers was determined by an atomic force microscopic analysis. The analysis of the mouse rhodopsin complex has enabled us to discuss the oligomerization based on structural data. Although many unsolved problems still remains, the idea that GPCRs directly interact to form oligomers has been gradually accepted. One of the recent findings in the GPCR investigations is the clarification of the mechanisms of GPCR oligomerization at a molecular level. Most of these studies have suggested the importance of transmembrane alpha-helices for GPCR oligomerization. In this review, we will first summarize the importance of GPCR oligomerization and the functions of GPCRs. Then, we will explain the involvement of transmembrane alpha-helices in the oligomerization and a drug design strategy that targets these regions for GPCR oligomerization. Considering the current drug design methods, which are based on the modification of the protein-protein interactions of soluble regions of proteins, a "peptide mimic approach" that targets the transmembrane alpha-helices constituting the interfaces would be promising in drug discovery for GPCR oligomerization. For that purpose, we must know the positions of the interfaces. However, problems specific to membrane proteins have made it difficult to identify the positions of the interfaces experimentally. Therefore, information about the interfaces predicted by bioinformatics approaches is valuable. At the end of this review, several bioinformatics approaches toward interface prediction for oligomerization are introduced. The benefits and the pitfalls of these approaches are also discussed.     

Proteome-wide classification and identification of mammalian-type GPCRs by binary topology pattern

G protein-coupled receptors (GPCRs), a large eukaryotic protein family, have proved difficult to comprehensively detect and functionally identify by homology searches and domain detection, because they are highly divergent and their sequences share strikingly little similarity. Transmembrane (TM) topology pattern analysis has been used to classify TM proteins, and such patterns are conserved within GPCRs of similar function. Here, we developed a stepwise binary topology pattern (BTP) method for GPCR classification and identification and used it to identify and classify mammalian-type GPCRs in the genomes of 10 different eukaryotic species. A binary topology pattern was obtained for each functional class or group by assigning binary loop threshold lengths of "0" (short loop) or "1" (long loop). The GPCR-classification ability of the BTP method had quite high accuracies for classifying GPCR functions at the class level (Classes A, B, C, Frizzled/Smoothened, Non-GPCR, based on the GPCRDB classification scheme), with many classes being classified with 100% accuracy. Sufficiently high accuracies were also maintained at the functional group level, 0.945 over 15 functional groups. Proteome-wide mammalian-type GPCR searches in 10 eukaryotic genomes (H. sapiens, M. musculus, F. rubripes, C. intestinalis, A. thaliana, D. melanogaster, A. gambiae, C. elegans, P. falciparum, S. cerevisiae) using the BTP method showed much higher classification/identification in non-mammalian genomes than typical BLAST searches, in which a higher number of sequences were classified as Non-GPCR. This stepwise BTP method should prove useful for the identification and functional classification of GPCRs from the genomes of a wide range of species.     

Direct interaction between an allosteric agonist pepducin and the chemokine receptor CXCR4

Cell surface heptahelical G protein-coupled receptors (GPCRs) mediate critical cellular signaling pathways and are important pharmaceutical drug targets. (1) In addition to traditional small-molecule approaches, lipopeptide-based GPCR-derived pepducins have emerged as a new class of pharmaceutical agents. (2, 3) To better understand how pepducins interact with targeted receptors, we developed a cell-based photo-cross-linking approach to study the interaction between the pepducin agonist ATI-2341 and its target receptor, chemokine C-X-C-type receptor 4 (CXCR4). A pepducin analogue, ATI-2766, formed a specific UV-light-dependent cross-link to CXCR4 and to mutants with truncations of the N-terminus, the known chemokine docking site. These results demonstrate that CXCR4 is the direct binding target of ATI-2341 and suggest a new mechanism for allosteric modulation of GPCR activity. Adaptation and application of our findings should prove useful in further understanding pepducin modulation of GPCRs as well as enable new experimental approaches to better understand GPCR signal transduction.     

Ligand and structure-based methodologies for the prediction of the activity of G protein-coupled receptor ligands

Accurate in silico models for the quantitative prediction of the activity of G protein-coupled receptor (GPCR) ligands would greatly facilitate the process of drug discovery and development. Several methodologies have been developed based on the properties of the ligands, the direct study of the receptor-ligand interactions, or a combination of both approaches. Ligand-based three-dimensional quantitative structure-activity relationships (3D-QSAR) techniques, not requiring knowledge of the receptor structure, have been historically the first to be applied to the prediction of the activity of GPCR ligands. They are generally endowed with robustness and good ranking ability; however they are highly dependent on training sets. Structure-based techniques generally do not provide the level of accuracy necessary to yield meaningful rankings when applied to GPCR homology models. However, they are essentially independent from training sets and have a sufficient level of accuracy to allow an effective discrimination between binders and nonbinders, thus qualifying as viable lead discovery tools. The combination of ligand and structure-based methodologies in the form of receptor-based 3D-QSAR and ligand and structure-based consensus models results in robust and accurate quantitative predictions. The contribution of the structure-based component to these combined approaches is expected to become more substantial and effective in the future, as more sophisticated scoring functions are developed and more detailed structural information on GPCRs is gathered.     

An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application

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. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.     

The use of immortalized cell lines in GPCR screening: the good, bad and ugly

For most membrane-bound molecular targets, including G protein linked receptors (GPCRs), the optimal approach in drug discovery involves the use of cell based high throughput screening (HTS) technologies to identify compounds that modulate target activity. Most GPCRs have been cloned and can therefore be routinely expressed in immortalized cell lines. These cells can be easily and rapidly grown in unlimited quantities making them ideal for use in current HTS technologies. A significant advantage of this approach is that immortalized recombinant cells provide a homogenous background for expression of the target which greatly facilitates consistency in screening, thus allowing for a better understanding of the mechanism of action of the interacting compound or drug. Nonetheless, it is now evident that numerous disparities exist between the physiological environment of screening systems using recombinant cells and natural tissues. This has lead to a problem in the validity of the pharmacological data obtained using immortalized cells in as much as such cells do not always reflect the desired clinical efficacy and safety of the compounds under examination. This brief review discusses these issues and describes how they influence the discovery of drugs using modern HTS.     

G protein betagamma subunits as targets for small molecule therapeutic development

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.     

Antibodies against G-protein coupled receptors: novel uses in screening and drug development

Antibodies are components of the body's humoral immune system that are generated in response to foreign pathogens. Modern biomedical research has employed these very specific and efficient molecules designed by nature in the diagnosis of diseases, localization of gene products as well as in the rapid screening of targets for drug discovery and testing. In addition, the introduction of antibodies with fluorescent or enzymatic tags has significantly contributed to advances in imaging and microarray technology, which are revolutionizing disease research and the search for effective therapeutics. More recently antibodies have been used in the isolation of dimeric G protein-coupled receptor (GPCR) complexes. In this review, we discuss antibodies as powerful research tools for studying GPCRs, and their potential to be developed as drugs themselves.     

SEC–MS as an Approach to Isolate and Directly Identifying Small Molecular GPCR–Ligands from Complex Mixtures Without Labeling

G protein coupled receptors (GPCRs) belong to the most successful targets in drug discovery. However, the development of assays with an appropriately labeled high affinity reporter compound is laborious. In the present study an MS-based binding assay is described using the rat histamine receptor 2 (rH2) as a model GPCR system. Instead of using a purified receptor it is demonstrated that it is possible to use an unpurified receptor to extract active compounds from a solution or small mixture of compounds. By using SEC it is possible to separate the bound ligand from the unbound ligand. The major advantage of this approach is that there is no labeling of ligands required (direct monitoring based on the appropriate m/z values).     

Discovery of novel regulatory peptides by reverse pharmacology: spotlight on chemerin and the RF-amide peptides metastin and QRFP

Reverse pharmacology is a screening technology that matches G protein-coupled receptors (GPCRs) with unknown cognate ligands in cell-based screening assays by detection of agonist-induced signaling pathways. One decade spent pursuing orphan GPCR screening by this technique assigned over 30 ligand/receptor pairs and revealed previously known or novel undescribed ligands, mostly of a peptidic nature. In this review, we describe the discovery, characterization of the structural composition, biological function, physiological role and therapeutic potential of three recently identified peptidic ligands. These are metastin, QRFP in a context of five RF-amide genes described in humans and the chemoattractant, chemerin. Metastin was initially characterized as a metastasis inhibitor. Investigations using ligand/receptor pairing revealed that metastin was involved in a variety of physiological processes, including endocrine function during pregnancy and gonad development. The novel RF-amide QRFP is implicated in food intake and aldosterone release from the adrenal cortex in the rat. Chemerin, first described as TIG2, is upregulated in tazarotene-treated psoriatic skin. By GPCR screening, bioactive chemerin was isolated from ovarial carcinoma fluid as well as hemofiltrate. Characterization as a chemoattractant for immature dendritic cells and analysis of the expression profile of metastin and its receptor suggested a physiological role of chemerin as a mediator of the immune response, inflammatory processes and bone development.