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In this study, we developed an in-line methodology that combines analytical with pharmacological techniques to characterize metabolites of human histamine H4 receptor (hH4R) ligands. Liquid chromatographic separation of metabolic mixtures is coupled to high-resolution fractionation into 96- or 384-well plates and directly followed by a cell-based reporter gene assay to measure receptor signaling. The complete methodology was designed, optimized, validated, and ultimately miniaturized into a high-density well plate format. Finally, the methodology was demonstrated in a metabolic profiling setting for three hH4R lead compounds and the drug clozapine. This new methodology comprises integrated analytical separations, mass spectrometry, and a cell-based signal transduction-driven reporter gene assay that enables the implementation of comprehensive metabolic profiling earlier in the drug discovery process. © 2012 Society for Laboratory Automation and Screening.

The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.

The recent crystal structure determinations of druggable class A G protein-coupled receptors (GPCRs) have opened up excellent opportunities in structure-based ligand discovery for this pharmaceutically important protein family. We have developed and validated a customized structure-based virtual fragment screening protocol against the recently determined human histamine H(1) receptor (H(1)R) crystal structure. The method combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤22 heavy atoms) H(1)R ligands with an exceptionally high hit rate of 73%. Of the 26 tested fragments, 19 compounds had affinities ranging from 10 μM to 6 nM. The current study shows the potential of in silico screening against GPCR crystal structures to explore novel, fragment-like GPCR ligand space.

The histamine H(1) receptor (H(1)R) is a key player in acute inflammatory responses. Antihistamines are widely used to relief the symptoms of allergic rhinitis by antagonizing histamine binding to the H(1)R, without possessing intrinsic activity in classical assays such as guinea pig ileum contraction assays or intracellular Ca(2+) mobilization. Overexpression of H(1)R in heterologous cell lines unmasked the capacity of this receptor to signal in a histamine-independent manner. Moreover, a recent screen of therapeutic and reference antagonists on these H(1)R-overexpressing cells revealed that the majority of these drugs are in fact inverse agonists, as they inhibit basal H(1)R activity. In this chapter, we describe several approaches to study H(1)R constitutive signaling that can be used to identify inverse agonists acting at this blockbuster target.

The histamine H(1) receptor (H(1)R) is a key player in acute inflammatory responses. Antihistamines are widely used to relief the symptoms of allergic rhinitis by antagonizing histamine binding to the H(1)R, without possessing intrinsic activity in classical assays such as guinea pig ileum contraction assays or intracellular Ca(2+) mobilization. Overexpression of H(1)R in heterologous cell lines unmasked the capacity of this receptor to signal in a histamine-independent manner. Moreover, a recent screen of therapeutic and reference antagonists on these H(1)R-overexpressing cells revealed that the majority of these drugs are in fact inverse agonists, as they inhibit basal H(1)R activity. In this chapter, we describe several approaches to study H(1)R constitutive signaling that can be used to identify inverse agonists acting at this blockbuster target.

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus that can have serious consequences in immunocompromised hosts. Interestingly, HCMV genome encodes for four viral G protein-coupled receptors (vGPCRs), namely, US27, US28, UL33, and UL78. Thus far, US28 and UL33 have been shown to activate signaling pathways in a ligand-independent manner. US28 is the best characterized vGPCR and has been shown to be potentially involved in the development of HCMV-related diseases. As such, detailed investigation of these viral GPCR is of importance in order to understand molecular events occurring during viral pathogenesis and the potential identification of novel therapeutic targets. Herewith, we describe several approaches to study these HCMV-encoded vGPCRs. Using molecular biology, tags can be introduced in the vGPCRs, which may facilitate the study of their protein expression with various techniques, such as microscopy, Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Furthermore, radioligand binding studies can be performed to screen for ligands for vGPCRs, but also to study kinetics of internalization. We also describe several signal transduction assays that can evaluate the signaling activity of these vGPCRs. In addition, we discuss different proliferation assays and an in vivo xenograft model that were used to identify the oncogenic potential of US28. The study of these vGPCRs in their viral context can be examined using recombinant HCMV strains generated by bacterial artificial chromosome mutagenesis. Finally, we show how these mutants can be used in several pharmacological and biochemical assays.

Human herpesviruses (HHVs) are widespread pathogens involved in proliferative diseases, inflammatory conditions, and cardiovascular diseases. During evolution, homologs of human chemokine receptors were integrated into the HHV genomes. In addition to binding endogenous chemokines, these viral G protein-coupled receptors (vGPCRs) have acquired the ability to signal in a constitutive manner. Ligand-induced and ligand-independent and autocrine and paracrine signaling properties of vGPCRs modify the functions of the expressing cells and lead to transformation and escape from immune surveillance. Furthermore, cross-talk or heterodimerization with endogenous chemokine receptors represent other ways for vGPCRs to modify intracellular signaling and cellular functions. As such, these viral receptors seem to play a prominent role during viral pathogenesis and life cycle and thus represent innovative antiviral therapies. Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics.

Human cytomegalovirus (HCMV) is a widely spread herpesvirus that can have serious consequences in immunocompromised hosts. Interestingly, HCMV genome encodes for four viral G protein-coupled receptors (vGPCRs), namely, US27, US28, UL33, and UL78. Thus far, US28 and UL33 have been shown to activate signaling pathways in a ligand-independent manner. US28 is the best characterized vGPCR and has been shown to be potentially involved in the development of HCMV-related diseases. As such, detailed investigation of these viral GPCR is of importance in order to understand molecular events occurring during viral pathogenesis and the potential identification of novel therapeutic targets. Herewith, we describe several approaches to study these HCMV-encoded vGPCRs. Using molecular biology, tags can be introduced in the vGPCRs, which may facilitate the study of their protein expression with various techniques, such as microscopy, Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry. Furthermore, radioligand binding studies can be performed to screen for ligands for vGPCRs, but also to study kinetics of internalization. We also describe several signal transduction assays that can evaluate the signaling activity of these vGPCRs. In addition, we discuss different proliferation assays and an in vivo xenograft model that were used to identify the oncogenic potential of US28. The study of these vGPCRs in their viral context can be examined using recombinant HCMV strains generated by bacterial artificial chromosome mutagenesis. Finally, we show how these mutants can be used in several pharmacological and biochemical assays.

The CC chemokine CCL14a is constitutively expressed in a large variety of tissues and its inactive proform CCL14a(1–74) circulates in high concentrations in plasma. CCL14a(1–74) is converted into CCL14a(9–74) by the proteases urokinase-type plasminogen activator and plasmin and is a highly active agonist for the chemokine receptors CCR1 and CCR5. In this study, a new CCL14a analog, CCL14a(12–74), was isolated from blood filtrate. To elucidate the functional role of the N terminus, a panel of N-terminally truncated CCL14a analogs were tested on the receptors CCR1 to CCR5 and on the human cytomegalovirus (HCMV)-encoded chemokine receptor US28. The rank order of binding affinity to these receptors and of the activation of CCR1 and CCR5-mediated intracellular Ca2+ concentration mobilization is CCL14a(6–74)

Several herpesviruses and poxviruses contain genes encoding for G protein- coupled receptor (GPCR) proteins that are expressed on the surface of infected host cells and/or the viral envelope. Most of these membrane-associated proteins display highest homology to the subfamily of chemokine receptors known to play a key role in the immune system. Virally encoded chemokine receptors have been modified through evolutionary selection both in chemokine binding profile and signaling capacity, ultimately resulting in immune evasion and cellular reprogramming in favor of viral survival and replication. Insight in the role of virally encoded GPCRs during the viral lifecycle may reveal their potential as future drug targets.

G-protein-coupled receptors encoded by herpesviruses such as EBV, HCMV and KSHV are very interesting illustrations of the (patho)physiological importance of constitutive GPCR activity. These viral proteins are expressed on the cell surface of infected cells and often constitutively activate a variety of G-proteins. For some virus-encoded GPCRs, the constitutive activity has been shown to occur in vivo, i.e., in infected cells. In this paper, we will review the occurrence of virus-encoded GPCRs and describe their known signaling properties. Moreover, we will also review the efforts, directed towards the discovery of small molecule antagonist, that so far have been mainly focused on the HCMV-encoded GPCR US28. This virus-encoded receptor might be involved in cardiovascular diseases and cancer and seems an interesting target for drug intervention.

Premi a l'excel·lència investigadora. Àmbit de les Ciències de la Salut. 2008 G-protein coupled receptors (GPCRs) respond to a chemically diverse plethora of signal transduction molecules. The notion that GPCRs also signal without an external chemical trigger, i.e. in a constitutive or spontaneous manner, resulted in a paradigm shift in the field of GPCR pharmacology. With the recognition of constitutive GPCR activity and the fact that GPCR binding and signaling can be strongly affected by a single point mutation, GPCR pharmacogenomics obtained a lot of attention. For a variety of GPCRs, point mutations have been convincingly linked to human disease. Mutations within conserved motifs, known to be involved in GPCR activation, might explain the properties of some naturally occurring constitutively active GPCR variants linked to disease. A brief history historical introduction to the present concept of constitutive receptor activity is given and the pharmacogenomic and the structural aspects of constitutive receptor activity are described.

Several herpesviruses encode G-protein-coupled receptor (vGPCR) proteins that are homologous to human chemokine receptors. In contrast to chemokine receptors, many vGPCRs signal in a ligand-independent (constitutive) manner. Such constitutive signaling is of major significance because various pathologies are associated with activating GPCR mutations. Constitutive activity of the human herpesvirus 8-encoded GPCR (ORF74), for example, is essential for its oncogenic potential to cause angioproliferative Kaposi's sarcoma-like lesions. The human cytomegalovirus (HCMV) encodes four GPCRs, of which US28 and UL33 display constitutive activity in transfected, but also HCMV-infected, cells. In addition, US28 is activated by a broad spectrum of chemokines. Furthermore, both US28 and UL33 show promiscuous G-protein coupling, whereas chemokine receptors activate primarily G i/o proteins. Thus, these vGPCRs are versatile signaling devices, reprogramming cellular signaling networks to modulate cellular function after infection. By these means, these HCMV-encoded receptors might contribute to HCMV-related pathologies.

G-protein coupled receptors encoded by viruses represent an unexplored class of potential drug targets. In this study, we describe the synthesis and pharmacological characterization of the first class of inverse agonists acting on the HCMV-encoded receptor US28. It is shown that replacement of the 4-hydroxy group of lead compound 1 with a methylamine group results in a significant 6-fold increase in affinity. Interestingly, increasing the rigidity of the spacer by the introduction of a double bond also leads to a significant increase in binding affinity compared to 1. These novel inverse agonists serve as valuable tools to elucidate the role of constitutive signaling in the pathogenesis of viral infection and may have therapeutic potential as leads for new antiviral drugs.

This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base substitutions/deletions/insertions that yield single/multiple amino acid substitutions/deletions/insertions and/or N- or C-terminal truncations in GPCRs. Polymerase chain reaction-based mutagenesis strategies allow substitutions/deletions/insertions of larger domains within GPCRs, creating truncated receptors or receptor chimeras. In addition, some guidelines are given and examples are provided to facilitate design and interpretation of mutational experiments.

This chapter describes three different strategies of receptor mutagenesis with their advantages, disadvantages, and limitations. Oligonucleotide-directed mutagenesis using either the Altered Sites II in vitro mutagenesis system or the GeneTailor site-directed mutagenesis system can generate base substitutions/deletions/insertions that yield single/multiple amino acid substitutions/deletions/insertions and/or N- or C-terminal truncations in GPCRs. Polymerase chain reaction-based mutagenesis strategies allow substitutions/deletions/insertions of larger domains within GPCRs, creating truncated receptors or receptor chimeras. In addition, some guidelines are given and examples are provided to facilitate design and interpretation of mutational experiments.

Mammalian gonadotropins are highly selective. Charge differences between the Cys(10-11) sequence of FSHbeta and LHbeta/CGbeta seat-belt loops determine the ability of these hormones to interact with the LH-R. Selective FSH-R binding is mainly dependent on the presence of an FSHbeta-specific sequence between Cys(11-12) of the seat-belt loop. Intriguingly, African catfish LHbeta (cfLHbeta) lacks a positively charged Cys(10-11) region and stimulates both catfish LH-R and FSH-R with comparable potencies. Our studies on the promiscuous behaviour of cfLH using chimeric gonadotropins revealed that the Cys(10-11) region of cfLHbeta contains cfLH-R-selective determinants, whereas the Cys(11-12) region of cfLHbeta confers FSH-R-stimulating activity to cfLH. Hence, the location of receptor-selective determinants appeared to be fairly well conserved throughout evolution, despite the low sequence identity between mammalian and catfish seat-belt loops. Moreover, various structure-function differences between gonadotropins are discussed in the context of the different (female) reproductive strategies between mammalian and non-mammalian species that required the divergence to a more specific LH-R-stimulating activity of one of the gonadotropins in mammals.

The difference in hormone selectivity between the human follicle-stimulating hormone receptor (hFSH-R) and human luteinizing hormone/chorionic gonadotropin receptor (hLH-R) is determined by their approximately 350 amino acid-long N-terminal receptor exodomains that allow the mutually exclusive binding of human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) when these hormones are present in physiological concentrations. The exodomains of each of these receptors consist of a nine-leucine-rich repeat-containing subdomain (LRR subdomain) flanked by N- and C-terminal cysteine-rich subdomains. Chimeric receptors, in which the structural subdomains of the hFSH-R exodomain were substituted with those of the hLH-R, showed a similar high responsiveness to human chorionic gonadotropin (hCG) and hLH as long as they harbored the LRR subdomain of the hLH-R. In addition, these chimeric receptors showed no responsiveness to hFSH. The LRR subdomains of the gonadotropin receptor exodomains are predicted to adopt a horseshoe-like conformation, of which the hormone-binding concave surface is composed of nine parallel beta-strands. Receptors in which individual beta-strands of the hFSH-R were replaced with the corresponding hLH-R sequences revealed that hCG and hLH selectivity is predominantly determined by hLH-R beta-strands 3 and 6. A mutant receptor in which the hFSH-R beta-strands 3 and 6 were substituted simultaneously with their hLH-R counterparts displayed a responsiveness to hCG and hLH similar to that of the wild type hLH-R. Responsiveness to hFSH was not affected by most beta-strand substitutions, suggesting the involvement of multiple low-impact determinants for this hormone.

The pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) regulate steroidogenesis and spermatogenesis by activating receptors expressed by Leydig cells (LH receptor) and Sertoli cells (FSH receptor), respectively. This concept is also valid in fish, although the piscine receptors may be less discriminatory than their mammalian counterparts. The main biological activity of LH is to regulate Leydig-cell steroid production. Steroidogenesis is moreover modulated in an autoregulatory manner by androgens. The male sex steroids (testosterone in higher vertebrates, 11-ketotestosterone in fish) are required for spermatogenesis, but their mode of action has remained obscure. While piscine FSH also appears to have steroidogenic activity, specific roles have not been described yet in the testis. The feedback of androgens on gonadotrophs presents a complex pattern. Aromatizable androgens/estrogens stimulate LH synthesis in juvenile fish; this effect fades out during maturation. This positive feedback on LH synthesis is balanced by a negative feedback on LH release, which may involve GnRH neurones. While the role of GnRH as LH secretagogue is evident, we have found no indication in adult male African catfish for a direct, GnRH-mediated stimulation of LH synthesis. The limited available information at present precludes a generalized view on the testicular feedback on FSH.

Chronic activation of Wnt/ß-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ß-catenin. In this study we show that this viral receptor constitutively activates ß-catenin and enhances ß-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ß-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ß-catenin. Moreover, cells infected with HCMV show significant increases in ß-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ß-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases. © 2012 Langemeijer et al.

In the article by Leurs et al. ‘En route to new blockbuster anti-histamines: surveying the offspring of the expanding histamine receptor family’, which was published in the April 2011 issue of Trends in Pharmacological Sciences, there are two errors in Figure 2. First, compound ADP-916 should be listed APD916. Second, the Thomson Reuters Pharma database should be listed as the source for the structures of compounds HPP404 and APD916 (both retrieved Sept. 2010). In the section ‘Narcolepsy’, compound ADP-916 should also be correctly named as APD916. Trends in Pharmacological Sciences and the authors apologize to the readers for these errors.

With the recognition of two new histamine receptors at the start of the new millennium, the field of histamine research has seen a clear revival. In the last 10 years, many academic and industrial groups have taken up the challenge to target these new members of the aminergic G-protein-coupled receptor (GPCR) family. Histamine receptor research nicely illustrates how GPCR research has changed in the post-genomic era. There is a growing understanding of GPCR structure, function and modulation at a molecular level. Emerging concepts such as receptor isoforms, GPCR oligomerization and ligand-biased signaling are all being studied, but their clinical relevance remains to be determined. The histamine H 3 and H 4 drug development programs can help to establish the link between these molecular features and clinical efficacy. Several new anti-histamines are now being tested for diverse clinical applications and are poised to become the next blockbuster drugs targeting histamine receptors.

US28 is a chemokine receptor encoded by the human cytomegalovirus (hCMV) and seems to be crucial for viral propagation. It is a constitutively active receptor. For instance it was shown that US28 signals via Gq/PLC and activates the transcription factors nuclear factor κB (NFκB), cyclic AMP response element binding protein (CREB) and nuclear factor of activated T-cells (NFAT). Hence, the receptor may play a key role in the early reprogramming of the host cells. Interestingly, US28 is constitutively phosphorylated and endocytosed. It has also been suggested that, during viral propagation, US28 is integrated into the viral envelope in the lysosomes of host cells. A candidate protein for sorting many G protein-coupled receptors to lysosomes is the G protein-coupled receptor-associated protein-1 (GASP-1). Here, we show that GASP-1 and the C-terminal part of GASP-1 (cGASP-1) interact with the far C-terminal end of US28 in vitro. We assessed this by using in vitro translated, [35S]methionine-radiolabelled GASP-1 and cGASP-1 and several different US28 C-terminal mutations and truncations in a GST pull-down assay.

Developments in G protein-coupled receptor (GPCR) structural biology provide insights into GPCR-ligand binding. Compound 1 (4-(2-benzylphenoxy)piperidine) with high ligand efficiency for the histamine H1 receptor (H1R) was used to design derivatives to investigate the roles of (i) the amine-binding region, (ii) the upper and lower aromatic region, and (iii) binding site solvation. SAR analysis showed that the amine-binding region serves as the primary binding hot spot, preferably binding small tertiary amines. In silico prediction of water network energetics and mutagenesis studies indicated that the displacement of a water molecule from the amine-binding region is most likely responsible for the increased affinity of the N-methylated analog of 1. Deconstruction of 1 showed that the lower aromatic region serves as a secondary binding hot spot. This study demonstrates that an X-ray structure in combination with tool compounds, assessment of water energetics, and mutagenesis studies enables SAR exploration ...