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The histamine H1 receptor (H1R) has recently been implicated in mediating cell proliferation and cancer progression; therefore, high-affinity H1R-selective fluorescent ligands are desirable tools for further investigation of this behavior in vitro and in vivo. We previously reported a H1R fluorescent ligand, bearing a peptide-linker, based on antagonist VUF13816 and sought to further explore structure-activity relationships (SARs) around the linker, orthostere, and fluorescent moieties. Here, we report a series of high-affinity H1R fluorescent ligands varying in peptide linker composition, orthosteric targeting moiety, and fluorophore. Incorporation of a boron-dipyrromethene (BODIPY) 630/650-based fluorophore conferred high binding affinity to our H1R fluorescent ligands, remarkably overriding the linker SAR observed in corresponding unlabeled congeners. Compound 31a, both potent and subtype-selective, enabled H1R visualization using confocal microscopy at a concentration of 10 nM. Molecular docking of 31a with the human H1R predicts that the optimized peptide linker makes interactions with key residues in the receptor.

The histamine H(3) receptor (H(3)R) represents a highly attractive drug target for the treatment of various central nervous system disorders, but the discovery of novel H(3)R targeting compounds relies on the assessment of highly amplified intracellular signaling events that do not only reflect H(3)R modulation and carry the risk of high false-positive and -negative screening rates. To address these limitations, we designed an intramolecular H(3)R biosensor based on the principle of bioluminescence resonance energy transfer (BRET) that reports the receptor's real-time conformational dynamics and provides an advanced tool to screen for both H(3)R agonists and inverse agonists in a live cell screening-compatible assay format. This conformational G-protein-coupled receptor (GPCR) sensor allowed us to characterize the pharmacological properties of known and new H(3) receptor ligands with unprecedented accuracy. Interestingly, we found that one newly developed H(3) receptor ligand possesses even stronger inverse agonistic activity than reference H(3)R inverse agonists including the current gold standard pitolisant. Taken together, we describe here the design and validation of the first screening-compatible H(3)R conformational biosensor that will aid in the discovery of novel H(3)R ligands and can be employed to gain deeper insights into the (in-)activation mechanism of this highly attractive drug target.

We report a detailed structure–activity relationship for the scaffold of VUF16216, a compound we have previously communicated as a small-molecule efficacy photoswitch for the peptidergic chemokine GPCR CXCR3. A series of photoswitchable azobenzene ligands was prepared through various synthetic strategies and multistep syntheses. Photochemical and pharmacological properties were used to guide the design iterations. Investigations of positional and substituent effects reveal that halogen substituents on the ortho-position of the outer ring are preferred for conferring partial agonism on the cis form of the ligands. This effect could be expanded by an electron-donating group on the para-position of the central ring. A variety of efficacy differences between the trans and cis forms emerges from these compounds. Tool compounds VUF15888 (4d) and VUF16620 (6e) represent more subtle efficacy switches, while VUF16216 (6f) displays the largest efficacy switch, from antagonism to full agonism. The compound class disclosed here can aid in new photopharmacology studies of CXCR3 signaling.

Drug-target binding kinetics are an important predictor of in vivo drug efficacy, yet the relationship between ligand structures and their binding kinetics is often poorly understood. We show that both rupatadine (1) and desloratadine (2) have a long residence time at the histamine H1 receptor (H1R). Through development of a [3H]levocetirizine radiolabel, we find that the residence time of 1 exceeds that of 2 more than 10-fold. This was further explored with 22 synthesized rupatadine and desloratadine analogues. Methylene-linked cycloaliphatic or β-branched substitutions of desloratadine increase the residence time at the H1R, conveying a longer duration of receptor antagonism. However, cycloaliphatic substituents directly attached to the piperidine amine (i.e., lacking the spacer) have decreased binding affinity and residence time compared to their methylene-linked structural analogues. Guided by docking studies, steric constraints within the binding pocket are hypothesized to explain the observed differences in affinity and binding kinetics between analogues.

The adhesion class B2 family of G protein-coupled receptors (GPCRs) plays a key role in important physiological processes and their dysfunction is linked to brain malformations and tumorigenesis. Although information regarding their signaling properties is starting to emerge, the structural motifs and interaction networks that determine 7 transmembrane (TM) signaling of class B2 GPCRs remain to be elucidated. Comparative sequence-structure analyses of class B2 GPCRs and the recently solved active class B1 structures show that class B2 GPCRs include different elements of the conserved residue motifs that determine class B1 activation. Combined site-directed mutagenesis and molecular dynamics studies were performed to give detailed insights into the role of 7TM interaction networks in signaling of two representative class B2 receptors, ADGRG1 (GPR56) and ADGRL4 (ELTD1). The systematic investigation of class B1/B2 sequence motifs provides consistent structure-function relationships that can be translated to the whole class B2 GPCR family and suggests that class B1 and B2 GPCRs share conserved intramolecular 7TM interactions. This improved understanding in adhesion GPCR structure and constitutive signaling can accelerate drug design campaigns for this chemically unexplored receptor class.

The Exome Aggregation Consortium has collected the protein-encoding DNA sequences of almost 61,000 unrelated humans. Analysis of this dataset for G protein-coupled receptor (GPCR) proteins (available at GPCRdb) revealed a total of 463 naturally occurring genetic missense variations in the histamine receptor family. In this research, we have analyzed the distribution of these missense variations in the four histamine receptor subtypes concerning structural segments and sites important for GPCR function. Four missense variants R1273.52×52H, R13934.57×57H, R4096.29×29H, and E4106.30×30 K, were selected for the histamine H1 receptor (H1R) that were hypothesized to affect receptor activity by interfering with the interaction pattern of the highly conserved D(E)RY motif, the so-called ionic lock. The E4106.30×30 K missense variant displays higher constitutive activity in G protein signaling as compared to wild-type H1R, whereas the opposite was observed for R1273.52×52H, R13934.57×57H, and R4096.29×29H. The E4106.30×30 K missense variant displays a higher affinity for the endogenous agonist histamine than wild-type H1R, whereas antagonist affinity was not affected. These data support the hypothesis that the E4106.30×30 K mutation shifts the equilibrium towards active conformations. The study of these selected missense variants gives additional insight into the structural basis of H1R activation and, moreover, highlights that missense variants can result in pharmacologically different behavior as compared to wild-type receptors and should consequently be considered in the drug discovery process.

Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

Despite the high diversity of histamine H3 receptor (H3R) antagonist/inverse agonist structures, partial or full H3R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H3R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H3R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 (14d) combines nanomolar on-target activity (pKi = 8.5, pEC50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H3R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H3R research.

Drug-target binding kinetics are suggested to be important parameters for the prediction of in vivo drug-efficacy. For G protein-coupled receptors (GPCRs), the binding kinetics of ligands are typically determined using association binding experiments in competition with radiolabelled probes, followed by analysis with the widely used competitive binding kinetics theory developed by Motulsky and Mahan. Despite this, the influence of the radioligand binding kinetics on the kinetic parameters derived for the ligands tested is often overlooked. To address this, binding rate constants for a series of histamine H1 receptor (H1R) antagonists were determined using radioligands with either slow (low koff) or fast (high koff) dissociation characteristics. A correlation was observed between the probe-specific datasets for the kinetic binding affinities, association rate constants and dissociation rate constants. However, the magnitude and accuracy of the binding rate constant-values was highly dependent on the used radioligand probe. Further analysis using recently developed fluorescent binding methods corroborates the finding that the Motulsky-Mahan methodology is limited by the employed assay conditions. The presented data suggest that kinetic parameters of GPCR ligands depend largely on the characteristics of the probe used and results should therefore be viewed within the experimental context and limitations of the applied methodology.

Glioblastoma (GBM) is the most aggressive and an incurable type of brain cancer. Human cytomegalovirus (HCMV) DNA and encoded proteins, including the chemokine receptor US28, have been detected in GBM tumors. US28 displays constitutive activity and is able to bind several human chemokines, leading to the activation of various proliferative and inflammatory signaling pathways. Here we show that HCMV, through the expression of US28, significantly enhanced the growth of 3D spheroids of U251− and neurospheres of primary glioblastoma cells. Moreover, US28 expression accelerated the growth of glioblastoma cells in an orthotopic intracranial GBM-model in mice. We developed highly potent and selective US28-targeting nanobodies, which bind to the extracellular domain of US28 and detect US28 in GBM tissue. The nanobodies inhibited chemokine binding and reduced the constitutive US28-mediated signaling with nanomolar potencies and significantly impaired HCMV/US28-mediated tumor growth in vitro and in vivo. This study emphasizes the oncomodulatory role of HCMV-encoded US28 and provides a potential therapeutic approach for HCMV-positive tumors using the nanobody technology.

WHIM syndrome is a rare congenital immunodeficiency disease, named after its main clinical manifestations: warts, hypogammaglobulinemia, infections, and myelokathexis, which refers to abnormal accumulation of mature neutrophils in the bone marrow. The disease is primarily caused by C-terminal truncation mutations of the chemokine receptor CXCR4, giving these CXCR4-WHIM mutants a gain of function in response to their ligand CXCL12. Considering the broad functions of CXCR4 in maintaining leukocyte homeostasis, patients are panleukopenic and display altered immune responses, likely as a consequence of impairment in the differentiation and trafficking of leukocytes. Treatment of WHIM patients currently consists of symptom relief, leading to unsatisfactory clinical responses. As an alternative and potentially more effective approach, we tested the potency and efficacy of CXCR4-specific nanobodies on inhibiting CXCR4-WHIM mutants. Nanobodies are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies. They combine the advantages of small-molecule drugs and antibody-based therapeutics due to their relative small size, high stability, and high affinity. We compared the potential of monovalent and bivalent CXCR4-specific nanobodies to inhibit CXCL12-induced CXCR4-WHIM-mediated signaling with the small-molecule clinical candidate AMD3100. The CXCR4-targeting nanobodies displace CXCL12 binding and bind CXCR4-wild type and CXCR4-WHIM (R334X/S338X) mutants and with (sub-) nanomolar affinities. The nanobodies' epitope was mapped to extracellular loop 2 of CXCR4, overlapping with the binding site of CXCL12. Monovalent, and in particular bivalent, nanobodies were more potent than AMD3100 in reducing CXCL12-mediated G protein activation. In addition, CXCR4-WHIM-dependent calcium flux and wound healing of human papillomavirus-immortalized cell lines in response to CXCL12 was effectively inhibited by the nanobodies. Based on these in vitro results, we conclude that CXCR4 nanobodies hold significant potential as alternative therapeutics for CXCR4-associated diseases such as WHIM syndrome.

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structure activity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions. (C) 2018 Elsevier Masson SAS. All rights reserved.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74.

Drug-target binding kinetics has recently attracted considerable interest in view of the potential predictive power for in vivo drug efficacy. The recently introduced antihistamine bilastine has a long duration of in vivo drug action, which outlasts pharmacological active bilastine concentrations in blood. To provide a molecular basis for the long duration of action, we explored the kinetics of bilastine binding to the human histamine H1 receptor using [3H]mepyramine binding studies and compared its pharmacodynamics properties to the reference compounds fexofenadine and diphenhydramine, which have a long (60 ± 20 min) and short (0.41 ± 0.1 min) residence time, respectively. Bilastine shows a long drug-target residence time at the H1 receptor (73 ± 5 min) and this results in a prolonged H1 receptor antagonism in vitro (Ca2+ mobilization in Fluo-4 loaded HeLa cells), following a washout of unbound antagonist. Hence, the long residence time of bilastine can explain the observed long duration of drug action in vivo.

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.

The increasing number of G protein-coupled receptor (GPCR) crystal structures offers new opportunities for histamine receptor homology modeling. However, computational prediction of ligand binding modes in GPCRs such as the histamine H4 receptor (H4R), a receptor that plays an important role in inflammation, remains a challenging task. In the current work we have combined complementary in silico receptor modeling approaches with in vitro ligand structure-activity relationship (SAR) and protein site-directed mutagenesis studies to elucidate the binding modes of different ligand classes in H4R. By systematically considering different H4R modelling templates, ligand binding poses, and ligand protonation states in combination with docking and MD simulations we are able to explain ligand-specific mutation effects and subtle differences in ligand SAR. Our studies confirm that a combined theoretical and experimental approach represents a powerful strategy to map ligand-protein interactions. © The Royal Society of Chemistry 2013.

Ligand residence time is thought to be a critical parameter for optimizing the in vivo efficacy of drug candidates. For the histamine H1 receptor (H1R) and other G protein-coupled receptors, the kinetics of ligand binding are typically measured by low throughput radioligand binding experiments using homogenized cell membranes expressing the target receptor. In this study, a real-time proximity assay between H1R and β-arrestin2 in living cells was established to investigate the dynamics of antihistamine binding to the H1R. No receptor reserve was found for the histamine-induced recruitment of β-arrestin2 to the H1R and the transiently recruited β-arrestin2 therefore reflected occupancy of the receptor by histamine. Antihistamines displayed similar kinetic signatures on antagonizing histamine-induced β-arrestin2 recruitment as compared to displacing radioligand binding from the H1R. This homogeneous functional method unambiguously determined the fifty-fold difference in the dissociation rate constant between mepyramine and the long residence time antihistamines levocetirizine and desloratadine.

Adhesion GPCRs (aGPCRs) form a subfamily of the large GPCR super family. Most aGPCRs are characterised by a non-covalent bipartite structure that consists of a large extracellular domain and a membrane-spanning 7 transmembrane domain. Typically, aGPCRs can combine cell adhesion by the large extracellular domain with intracellular signalling by the 7 transmembrane domain. Immune responses rely on cellular communication and subsequent defence reactions. Indeed, aGPCR ADGRB1 and members of the ADGRE class have been linked to processes like phagocytosis, leucocyte activation and migration. Nevertheless, research is hampered by absence of endogenous ligands, unknown activity of generated antibodies and non-identified signalling pathways. Yet, based on their membrane localisation and important function, aGPCRs could be novel drug targets to modulate leucocyte function.

After the recent description of β-arrestin2 recruitment to the human histamine H4 receptor (hH4R) in response to the well known H4R antagonist 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methyl-piperazine (JNJ 7777120), we evaluated in this study the efficacy of 31 known hH4R ligands to induce Gαi protein signaling and β-arrestin2 recruitment by the hH4R. The selected hH4R ligands belong to nine different structural classes that partly cover (pre)clinical trial candidates. We have identified hH4R ligands with a significant bias for the Gαi protein or β-arrestin2 pathway on the basis of efficacy differences. In addition, hH4R antagonists that did not show positive efficacy in either functional readouts were found. A common trend in pathway preference for the nine different ligand classes could not be observed. In particular, the isothiourea class shows very diverse results, varying from Gαi protein-biased or β-arrestin2-biased to nonbiased antagonists upon minor structural changes. The identified biased hH4R ligands are important pharmacological tools to unravel the significance of biased hH4R signaling in H4R pharmacology.

Most cells express a panel of different G protein-coupled receptors (GPCRs) allowing them to respond to at least a corresponding variety of extracellular ligands. In order to come to an integrative well-balanced functional response these ligand-receptor pairs can often cross-regulate each other. Although most GPCRs are fully capable to induce intracellular signalling upon agonist binding on their own, many GPCRs, if not all, appear to exist and function in homomeric and/or heteromeric assemblies for at least some time. Such heteromeric organization offers unique allosteric control of receptor pharmacology and function between the protomers and might even unmask 'new' features. However, it is important to realize that some functional consequences that are proposed to originate from heteromeric receptor interactions may also be observed due to intracellular crosstalk between signalling pathways of non-associated GPCRs. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3beta (GSK-3beta) function, promoted accumulation of beta-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells.

Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

Human herpesviruses are widespread infectious pathogens that have been associated with proliferative and inflammatory diseases. During viral evolution, human herpesviruses have pirated genes encoding viral G protein-coupled receptors (vGPCRs), which are expressed on infected host cells. These vGPCRs show highest homology to human chemokine receptors, which play a key role in the immune system. Importantly, vGPCRs have acquired unique properties such as constitutive activity and the ability to bind a broad range of human chemokines. This allows vGPCRs to hijack human proteins and modulate cellular signaling for the benefit of the virus, ultimately resulting in immune evasion and viral dissemination to establish a widespread and lifelong infection. Knowledge on the mechanisms by which herpesviruses reprogram cellular signaling might provide insight in the contribution of vGPCRs to viral survival and herpesvirus-associated pathologies.

The human herpes virus 6 (HHV-6)-encoded chemokine receptor U51 constitutively activates phospholipase C (PLC) and inhibits cAMP-responsive element (CRE)-mediated gene transcription via the activation of G(q/11) proteins. Yet, chemokines known to bind U51 differentially regulate U51 coupling to G proteins. CCL5/RANTES induced pertussis toxin (PTX)-insensitive increases in PLC activity and changes in intracellular free calcium concentration ([Ca2+]i), whereas both CCL2/MCP-1 and CCL11/eotaxin failed to stimulate PLC activity or increase [Ca2+]i. In contrast, all three chemokines counteracted the effects of U51 on CRE activity via the activation of PTX-sensitive G(i/o) proteins. For each of the tested chemokines, coexpression of U51 with a variety of G alpha subunits, however, revealed a distinct profile for preferred G-protein coupling, which could be shifted by modulation of the relative expression of G proteins. These findings are consistent with a chemokine-selective trafficking of receptor stimulus to distinct G proteins and suggest that the constitutive activity of U51 and the chemokine-induced signaling involve different active states of the receptor. By virtue of its ability to constitutively activate signaling pathways, its G-protein promiscuity, and the chemokine-directed trafficking of receptor stimulus, U51 can be considered a sensitive and versatile virally encoded signaling device, potentially of importance in HHV-6-related pathologies.

Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human chemokine receptor CCR1 and binds several chemokines of the CC family as well as the CX3C chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of chemokine dimerization and interaction with glycosaminoglycans (GAGs) on chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.

The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (Kd or Ki value) or target modulation (IC50). However, these metrics are determined at equilibrium conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the in vivo efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H1 receptor (H1R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H1R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H1R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the receptor recovery time. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H1R residence times, as determined with competitive radioligand binding experiments to the H1R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H1R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules.

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric Gi proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate Gi signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the Gi inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive Gi proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways. Background: CCX-CKR is considered to be a chemokine decoy receptor that is unable to signal. Results: Chemokines induce β-arrestin recruitment to CCX-CKR and pertussis toxin (PTX)-dependent CRE activity. Conclusion: PTX-sensitive G proteins hinder CCX-CKR coupling to other G proteins and consequently keep receptors silent. Significance: Recruitment of β-arrestin to CCX-CKR requests re-evaluation of the signaling capacity of this atypical receptor.

The chemokine receptor CXCR3 is a G-protein-coupled receptor that signals through the Gα(i) class of heterotrimeric G-proteins. CXCR3 is highly expressed on activated T cells and has been proposed to be a therapeutic target in autoimmune disease. CXCR3 is activated by the chemokines CXCL9, CXCL10 and CXCL11. CXCR3 signaling properties in response to CXCL10, CXCL11 and the synthetic agonist VUF10661 have previously been evaluated using conventional endpoint assays. In the present study, label-free impedance measurements were used to characterize holistic responses of CXCR3-expressing cells to stimulation with chemokines and VUF10661 in real time and to compare these responses with both G-protein and non-G-protein (β-arrestin2) mediated responses. Differences in response kinetics were apparent between the chemokines and VUF10661. Moreover, CXCR3-independent effects could be distinguished from CXCR3-specific responses with the use of the selective CXCR3 antagonist NBI-74330 and the Gα(i) inhibitor pertussis toxin. By comparing the various responses, we observed that CXCL9 is a biased CXCR3 agonist, stimulating solely G-protein-dependent pathways. Moreover, CXCR3-mediated changes in cellular impedance correlated with G-protein signaling, but not β-arrestin2 recruitment.

Contains fulltext : 103510.pdf (Publisher’s version ) (Closed access) We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the beta-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.

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 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.

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.

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)

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.

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.

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.

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 ...