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.     

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 throughput screening for orphan and liganded GPCRs

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Profiling of multiple signal pathway activities by multiplexing antibody and GFP-based translocation assays

Multiplexing of GFP based and immunofluorescence translocation assays enables easy acquisition of multiple readouts from the same cell in a single assay run. Immunofluorescence assays monitor translocation, phosphorylation, and up/down regulation of endogenous proteins. GFP-based assays monitor translocation of stably expressed GFP-fusion proteins. Such assays may be multiplexed along (vertical), across (horizontal), and between (branch) signal pathways. Examples of these strategies are presented: 1) The MK2-GFP assay monitors translocation of MK2-GFP from the nucleus to the cytoplasm in response to stimulation of the p38 pathway. By applying different immunofluorescent assays to the MK2 assay, a multiplexed HCA system is created for deconvolution of p38 pathway activation including assay readouts for MK2, p38, NFkappaB, and c-Jun. 2) A method for evaluating GPCR activation and internalization in a single assay run has been established by multiplexing GFP-based internalization assays with immunofluorescence assays for downstream transducers of GPCR activity: pCREB (cAMP sensor), NFATc1 (Ca(2+) sensor), and ERK (G-protein activation). Activation of the AT1 receptor is given as an example. 3) Cell toxicity readouts can be linked to primary readouts of interest via acquisition of secondary parameters describing cellular morphology. This approach is used to flag cytotoxic compounds and deselect false positives. The ATF6 Redistribution assay is provided as an example. These multiplex strategies provide a unique opportunity to enhance HCA data quality and save time during drug discovery. From a single assay run, several assay readouts are obtained that help the user to deconvolute the mode of action of test compounds.     

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.     

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).     

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.     

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.     

Chemogenomics and parasitology: small molecules and cell-based assays to study infectious processes

Infectious diseases caused by protozoan parasites--malaria, sleeping sickness, leishmaniasis, Chagas' disease, toxoplasmosis--remain chronic problems for humanity. We lack vaccines and have limited drug options effective against protozoa. Research into anti-protozoan drugs has accelerated with improved in vitro cultivation methods, enhanced genetic accessibility, completed genome sequences for key protozoa, and increased prominence of protozoan diseases on the agendas of well-resourced public figures and foundations. Concurrent advances in high-throughput screening (HTS) technologies and availability of diverse small molecule libraries offer the promise of accelerated discovery of new drug targets and new drugs that will reduce disease burdens imposed on humanity by parasitic protozoa. We provide a status report on HTS technologies in hand and cell-based assays under development for biological investigations and drug discovery directed toward the three best-characterized parasitic protozoa: Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. We emphasize cell growth assays and new insights into parasite cell biology speeding development of better cell-based assays, useful in primary screens for anti-protozoan drug leads and secondary screens to decipher mechanisms of action of leads identified in growth assays. Small molecules that interfere with specific aspects of protozoan biology, identified in such screens, will be valuable tools for dissecting parasite cell biology and developing anti-protozoan drugs. We discuss potential impacts on drug development of new consortia among academic, corporate, and public partners committed to discovery of new, effective anti-protozoan drugs.     

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.     

Photoswitching the Efficacy of a Small‐Molecule Ligand for a Peptidergic GPCR: from Antagonism to Agonism

For optical control of GPCR function, we set out to develop small‐molecule ligands with photoswitchable efficacy in which both configurations bind the target protein but exert distinct pharmacological effects, that is, stimulate or antagonize GPCR activation. Our design was based on a previously identified efficacy hotspot for the peptidergic chemokine receptor CXCR3 and resulted in the synthesis and characterization of five new azobenzene‐containing CXCR3 ligands. G protein activation assays and real‐time electrophysiology experiments demonstrated photoswitching from antagonism to partial agonism and even to full agonism (compound VUF16216). SAR evaluation suggests that the size and electron‐donating properties of the substituents on the inner aromatic ring are important for the efficacy photoswitching. These compounds are the first GPCR azo ligands with a nearly full efficacy photoswitch and may become valuable pharmacological tools for the optical control of peptidergic GPCR signaling.     

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.     

Biological applications of the receptor mimetic peptide mastoparan

The receptor mimetic and mast cell degranulating peptide mastoparan (MP) translocates cell membranes as an amphipathic alpha-helix, a feature that is undoubtedly a major determinant of bioactivity through the activation of heterotrimeric G proteins. Chimeric combinations of MP with G protein-coupled receptor (GPCR) ligands has produced peptides that exhibit biological activities distinct from their composite components. Thus, chimeric peptides such as galparan and M391 differentially modulate GTPase activity, display altered binding affinities for appropriate GPCRs and possess disparate secretory properties. MP and MP-containing chimerae also bind and modulate the activities of various other intracellular protein targets and are valuable tools to manipulate and study enzymatic activity, calcium homeostasis and apoptotic signalling pathways. In addition, charge delocalisation within the hydrophilic face of MP has produced analogues, including [Lys5, Lys8,Aib10]MP, that differentially regulate mast cell secretion and/or cytotoxicity. Finally, the identification of cell penetrant variants of MP chimerae has enabled the effective intracellular delivery of non-permeable biomolecules and presents an opportunity to target novel intracellular therapeutic loci.