Biomedical subjects

Rob Leurs

Publications and source records attributed to Rob Leurs.

At least 55 records · Page 3Linked to original sources

PubMed2004

Effect of the histamine H3-antagonist clobenpropit on spatial memory deficits induced by MK-801 as evaluated by radial maze in Sprague-Dawley rats.

This study was performed to investigate whether or not the histamine H3-antagonist clobenpropit can ameliorate spatial memory deficits induced by MK-801 (0.3 microg per site) as evaluated by an eight-arm radial maze task of rats. A bilateral intrahippocampal (i.h.) injection of clobenpropit (5, 10 microg per site, dose-dependent) markedly improved the working and reference memory deficits induced by MK-801. Its ameliorating effect was potentiated by histidine, but completely antagonized by immepip (2.5 microg per site), a selective H3-agonist. alpha-Fluoromethylhistidine (FMH, 25 microg per site), a selective histidine decarboxylase inhibitor prevented the ameliorating effect of clobenpropit on the working memory deficits induced by MK-801. In addition, the H(1-antagonist pyrilamine, but not the H2-antagonist cimetidine, also inhibited the procognitive effects of clobenpropit. Both FMH and pyrilamine did not significantly modulate the effect of clobenpropit on reference memory. Therefore, the results of this study suggest that the procognitive effects of clobenpropit in MK-801-induced working memory deficits is mediated by increasing endogenous histamine release. In addition, the ameliorating effect of clobenpropit on reference memory might be due to the increased release of neurotransmitters other than histamine.

Analysis of Variance↗

PubMed2004

Mutational analysis of the histamine H1-receptor binding pocket of histaprodifens.

Histaprodifens constitute a new class of histamine H(1)-receptor agonists. These ligands can be regarded as hybrid molecules, consisting of a histamine moiety linked at the two-position of the imidazole ring by a propyl chain to two phenyl rings, one of the characteristic features of several H(1)-receptor antagonists. To delineate the binding site of various histaprodifen-like ligands, we generated mutant histamine H(1) receptors, in which various amino acids, involved in the binding of either histamine or H(1)-receptor antagonists, were replaced by alanine. Wild-type and mutant H(1) receptors were transiently expressed in African green monkey kidney cells (COS-7) and evaluated for their interaction with histamine and various histaprodifens by [(3)H]mepyramine radioligand-binding studies and by nuclear factor kappaB (NF-kappaB) reporter-gene assays. Our data show that, within the histamine H(1)-receptor binding pocket, histaprodifens interact with both agonist and antagonist binding sites, resulting in high affinity histamine H(1)-receptor agonists.

Animals↗

PubMed2004

Furin is a chemokine-modifying enzyme: in vitro and in vivo processing of CXCL10 generates a C-terminally truncated chemokine retaining full activity.

Chemokines comprise a class of structurally related proteins that are involved in many aspects of leukocyte migration under basal and inflammatory conditions. In addition to the large number of genes, limited processing of these proteins by a variety of enzymes enhances the complexity of the total spectrum of chemokine variants. We have recently shown that the native chemokine CXCL10 is processed at the C terminus, thereby shedding the last four amino acids. The present study was performed to elucidate the mechanism in vivo and in vitro and to study the biological activity of this novel isoform of CXCL10. Using a combination of protein purification and mass spectrometric techniques, we show that the production of C-terminally truncated CXCL10 by primary keratinocytes is inhibited in vivo by a specific inhibitor of pro-protein convertases (e.g. furin) but not by inhibition of matrix metalloproteinases. Moreover, CXCL10 is processed by furin in vitro, which is abrogated by a mutation in the furin recognition site. Using GTPgammaS binding, Ca(2+) mobilization, and chemotaxis assays, we demonstrate that the C-terminally truncated CXCL10 variant is a potent ligand for CXCR3. Moreover, the inverse agonist activity on the virally encoded receptor ORF74 and the direct antibacterial activity of CXCL10 are fully retained. Hence, we have identified furin as a novel chemokine-modifying enzyme in vitro and most probably also in vivo, generating a C-terminally truncated CXCL10, which fully retains its (inverse) agonistic properties.

Amino Acid Sequence↗

PubMed2004

Role of H3-receptor-mediated signaling in anxiety and cognition in wild-type and Apoe-/- mice.

Increasing evidence supports a role for histamine as a neurotransmitter and neuromodulator in emotion and cognition. The H(3) receptor was first characterized as an autoreceptor that modulates histamine release and synthesis via negative feedback. Mice deficient in apoE (Apoe(-/-)) have been used to define the role of apoE in brain function. In the present study, we investigated the possible role of histamine H(3)-receptor-mediated signaling in anxiety and cognition in mice Apoe(-/-) and wild-type mice. H(3) antagonists increased measures of anxiety in wild-type, but not Apoe(-/-), mice. In contrast, H(3) antagonists similarly impaired object recognition in wild-type and Apoe(-/-) mice. In Apoe(-/-) mice, reduced negative feedback via H(3) receptors could contribute to increased signaling of H(1) receptors. Apoe(-/-) mice showed higher sensitivity to the anxiety-reducing effects of the H(1) receptor antagonist mepyramine than wild-type mice. These effects were dissociated from effects of mepyramine on the HPA axis. Compared to saline controls, mepyramine reduced plasma ACTH and corticosterone levels in wild-type, but not Apoe(-/-), mice. These data support a role for apoE in H(3) receptor signaling. H(3) antagonists were proposed as a treatment for cognitive disorders such as Alzheimer's disease, which is associated with increased anxiety and cognitive impairments. As H(3) antagonists increase measures of anxiety and impair object recognition in wild-type mice, the use of H(3) antagonists in cognitive disorders may be counterproductive and should be carefully evaluated.

Animals↗

PubMed2004

Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling.

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130-132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N(130)) to aspartic acid resulted in a mutant (N(130)D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N(130) is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N(130)A) was severely impaired in G(q/11)-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in G(i/0)-mediated signalling. Another protein that contained a modified NRY motif, R(131)A, did not show constitutive activity, whereas mutants Y(132)F and Y(132)A displayed similar activities to the WT receptor. This indicated that residue R(131) is critical for pR33 function in vitro, whereas Y(132) is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.

Amino Acid Motifs↗

PubMed2004

Large-scale overproduction, functional purification and ligand affinities of the His-tagged human histamine H1 receptor.

This report describes an efficient strategy for amplified functional purification of the human H1 receptor after heterologous expression in Sf9 cells. The cDNA encoding a C-terminally histidine-tagged (10xHis) human histamine H1 receptor was used to generate recombinant baculovirus in a Spodoptera frugiperda-derived cell line (IPLB-Sf9). As judged from its ligand affinity profile, functional receptor could be expressed at high levels (30-40 pmol per 10(6) cells). Rapid proteolysis in the cell culture led to limited fragmentation, without loss of ligand binding, but could be efficiently suppressed by including the protease inhibitor leupeptin during cell culture and all subsequent manipulations. Effective solubilization of functional receptor with optimal recovery and stability required the use of dodecylmaltoside as a detergent in the presence of a high concentration of NaCl and of a suitable inverse agonist. Efficient purification of solubilized receptor could be achieved by affinity chromatography over nickel(II) nitrilotriacetic acid resin. Functional membrane reconstitution of purified H1 receptor was accomplished in mixed soybean lipids (asolectin). The final proteoliposomic H1 receptor preparation has a purity greater than 90% on a protein basis and displays a ligand binding affinity profile very similar to the untagged receptor expressed in COS-7 cells. In conclusion, we are able to produce pharmacologically viable H1 receptor in a stable membrane environment allowing economic large-batch operation. This opens the way to detailed studies of structure-function relationships of this medically and biologically important receptor protein by 3D-crystallography, FT-IR spectroscopy and solid-state NMR spectroscopy.

Animals↗

PubMed2004

Differential activation of murine herpesvirus 68- and Kaposi's sarcoma-associated herpesvirus-encoded ORF74 G protein-coupled receptors by human and murine chemokines.

Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated herpesvirus [KSHV]). The open reading frame ORF74 of HVS, KSHV, and MHV-68 encodes a protein with homology to G protein-coupled receptors and chemokine receptors in particular. ORF74 of KSHV (human ORF74 [hORF74]) is highly constitutively active and has been implicated in the pathogenesis of Kaposi's sarcoma. MHV-68-encoded ORF74 (mORF74) is oncogenic and has been implicated in viral replication and reactivation from latency. Here, we show that mORF74 is a functional chemokine receptor. Chemokines with an N-terminal glutamic acid-leucine-arginine (ELR) motif (e.g., KC and macrophage inflammatory protein 2) act as agonists on mORF74, activating phospholipase C, NF-kappaB, p44/p42 mitogen-activated protein kinase, and Akt signaling pathways and inhibiting formation of cyclic AMP. Using (125)I-labeled CXCL1/growth-related oncogene alpha as a tracer, we show that murine CXCL10/gamma interferon-inducible protein 10 binds mORF74, and functional assays show that it behaves as an antagonist for this virally encoded G protein-coupled receptor. Profound differences in the upstream activation of signal transduction pathways between mORF74 and hORF74 were found. Moreover, in contrast to hORF74, no constitutive activity of mORF74 could be detected.

Animals↗

PubMed2003

Synthesis and pharmacological identification of neutral histamine H1-receptor antagonists.

In the present study we searched for neutral antagonists for the human histamine H(1)-receptor (H(1)R) by screening newly synthesized ligands that are structurally related to H(1)R agonists for their affinity using radioligand displacement studies and by assessing their functional activity via performing a NF-kappaB driven reporter-gene assay that allows for the detection of both agonistic and inverse agonistic responses. Starting from the endogenous agonist for the H(1)R, histamine, we synthesized and tested various analogues and ultimately identified several compounds with partial inverse agonistic properties and two neutral H(1)-receptor antagonists, namely 2-[2-(4,4-diphenylbutyl)-1H-imidazol-4-yl]ethylamine (histabudifen, 18d) (pK(i) = 5.8, alpha = 0.02) and 2-[2-(5,5-diphenylpentyl)-1H-imidazol-4-yl]ethylamine (histapendifen, 18e) (pK(i) = 5.9, alpha = -0.09).

Animals↗

PubMed2003

Effects of clobenpropit on pentylenetetrazole-kindled seizures in rats.

The purpose of this study was to investigate whether or not clobenpropit, a selective and potent histamine H(3) receptor antagonist, can protect from pentylenetetrazole (35 mg/kg)-kindled seizures in rats. I.c.v. injection with clobenpropit (10 and 20 microg) significantly delayed the seizure stage and prolonged the latency to the onset of myoclonic jerks and the latency to the clonic generalized seizure in a dose-dependent manner. The protection by clobenpropit (20 microg) was completely antagonized by both immepip (5 and 10 microg, i.c.v.), a selective potent histamine H(3) receptor agonist, and alpha-fluoromethylhistidine (alpha-FMH, 50 microg, i.c.v.), a selective histidine decarboxylase inhibitor. In addition, clobenpropit markedly potentiated the histidine (100 and 200 mg/kg)-induced inhibition of pentylenetetrazole-kindled seizures. Pyrilamine (2 and 5 microg, i.c.v.) reversed the inhibition of pentylenetetrazole-kindled seizures induced by clobenpropit, whereas cimetidine had no effect even at a high dose of 5 microg. These results indicate that clobenpropit protects against pentylenetetrazole-kindled seizures in rats, and that its action is mainly due to the activation of endogenous histamine by blocking autoinhibitory presynaptic histamine H(3) receptors.

Animals↗

PubMed2003

Synthesis and structure-activity relationships of conformationally constrained histamine H(3) receptor agonists.

Immepip, a conformationally constrained analogue of the histamine congener imbutamine, shows high affinity and functional activity on the human H(3) receptor. Using histamine and its homologues as prototypes, other rigid analogues containing either a piperidine or pyrrolidine ring in the side chain were synthesized and tested for their activities at the human H(3) receptor and the closely related H(4) receptor. In the series of piperidine containing analogues, immepip was found to be the most potent H(3) receptor agonist, whereas its propylene analogue 13a was identified as a high-affinity neutral antagonist for the human H(3) receptor. Moreover, replacement of the piperidine ring of immepip by a pyrrolidine ring led to a pair of enantiomers that show a distinct stereoselectivity at the human H(3) and H(4) receptor.

Binding, Competitive↗

PubMed2003

Constitutively active Gq/11-coupled receptors enable signaling by co-expressed G(i/o)-coupled receptors.

Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.

Animals↗

PubMed2003

Constitutive signaling of the human cytomegalovirus-encoded receptor UL33 differs from that of its rat cytomegalovirus homolog R33 by promiscuous activation of G proteins of the Gq, Gi, and Gs classes.

The human cytomegalovirus (HCMV) UL33 gene is conserved among all beta-herpesviruses and encodes a protein that shows sequence similarity with chemokine receptors belonging to the family of G protein-coupled receptors. Here, we show that HCMV UL33 is predominantly transcribed as a spliced mRNA of which the 5' terminus is localized 55 bp upstream of the start codon. Like its homolog from rat cytomegalovirus (RCMV), R33, UL33 activates multiple signaling pathways in a ligand-independent manner. Although both receptors constitutively activate phospholipase C via G(q/11), and partially via G(i/o)-mediated pathways, they exhibit profound differences in the modulation of cAMP-responsive element (CRE) activation. R33 constitutively inhibits, whereas UL33 constitutively enhances CRE-mediated transcription. For R33, the inhibition of CRE-driven transcription is entirely G(i/o)-mediated. For UL33, however, CRE-mediated transcription is modulated not only through coupling to Galpha(i/o) but also through coupling to Galphas. In addition, UL33 was found to enhance CRE activation through the Rho/p38 pathway, via Gbetagamma. Interestingly, by studying chimeric UL33/R33 proteins, we found the C-terminal cytoplasmic tail of UL33, but not that of R33, to be responsible for the activation of G(i/o) proteins. A UL33-deficient variant of HCMV was generated to analyze UL33-signaling properties in a physiologically relevant model system. Data obtained with infected cells show that HCMV induces CRE activation, and this effect is, at least in part, dependent on UL33 expression. Taken together, our data indicate that constitutive signaling of UL33 differs from that of R33 by promiscuous activation of G proteins of the Gq, G(i/o), as well as Gs class. Thus, HCMV may effectively use UL33 to orchestrate multiple signaling networks within infected cells.

Amino Acid Sequence↗

PubMed2003

A selective human H(4)-receptor agonist: (-)-2-cyano-1-methyl-3-[(2R,5R)-5- [1H-imidazol-4(5)-yl]tetrahydrofuran-2-y] methylguanidine.

A series of 16 compounds related to chiral 4(5)-(5-aminomethyltetrahydrofuran-2-yl)imidazoles (1) have been designed, synthesized, and examined in vitro by radioligand displacement studies and functional assays for both the human H(3)- and H(4)-receptors expressed in SK-N-MC cells. Among them, the (2S,5S)-isomer 1d of amino compounds showed approximately 300-fold higher selectivity at the H(3)-receptor than the H(4)-receptor. On the other hand, (2R,5S)- and (2R,5R)-cyanoguanidines 3b and 3c, in which the amino group of the compounds 1b and 1c was substituted by the cyanoguanidino moiety, bound to the H(4)-receptor with a pEC(50) value of 6.65 and 7.11, respectively, and had >40-fold selectivities over the H(3)-receptor. As such, 3b and 3c are the first selective H(4) receptor agonists.

Binding, Competitive↗

PubMed2003

Improgan antinociception does not require neuronal histamine or histamine receptors.

Improgan, a chemical congener of the H(2) antagonist cimetidine, induces antinociception following intracerebroventricular (i.c.v.) administration in rodents, but the mechanism of action of this compound remains unknown. Because the chemical structure of improgan closely resembles those of histamine and certain histamine blockers, and because neuronal histamine is known to participate in pain-relieving responses, the antinociceptive actions of improgan were evaluated in mice containing null mutations in the genes for three histamine receptors (H(1), H(2), and H(3)) and also in the gene for histidine decarboxylase (the histamine biosynthetic enzyme). Similar to earlier findings in Swiss-Webster mice, improgan induced maximal, reversible, dose-related reductions in thermal nociceptive responses in ICR mice, but neither pre-improgan (baseline) nor post-improgan nociceptive latencies were changed in any of the mutant mice as compared with wild-type controls. Improgan also had weak inhibitory activity in vitro (pK(i)=4.7-4.9) on specific binding to three recently-discovered, recombinant isoforms of the rat H(3) receptor (H(3A), H(3B), and H(3C)). The present findings strongly support the hypothesis that neuronal histamine and its receptors fail to play a role in improgan-induced antinociception.

Analgesics↗

PubMed2003

CXCR3-mediated chemotaxis of human T cells is regulated by a Gi- and phospholipase C-dependent pathway and not via activation of MEK/p44/p42 MAPK nor Akt/PI-3 kinase.

The chemokines CXCL9, 10, and 11 exert their action via CXC chemokine receptor-3 (CXCR3), a receptor highly expressed on activated T cells. These interferon gamma (IFNgamma)-induced chemokines are thought to be crucial in directing activated T cells to sites of inflammation. As such, they play an important role in several chronic inflammatory diseases including ulcerative colitis, multiple sclerosis, artherosclerosis, and delayed-type hypersensitivity reactions of the skin. In this study, we first demonstrate that in COS-7 cells heterologously expressing CXCR3, CXCL11 is a potent activator of the pertussis toxin (PTX)-sensitive p44/p42 mitogen-activated protein kinase (MAPK) and Akt/phosphatidylinositol 3 kinase (PI3K) pathways. Next, we show that these signal transduction pathways are also operative and PTX sensitive in primary human T cells expressing CXCR3. Importantly, abrogation of these signaling cascades by specific inhibitors did not block the migration of T cells toward CXCR3 ligands, suggesting that MAPK and Akt activation is not crucial for CXCR3-mediated chemotaxis of T cells. Finally, we demonstrate that CXCR3-targeting chemokines control T-cell migration via PTX-sensitive, phospholipase C pathways and phosphatidylinositol kinases other than class I PI3Kgamma.

Animals↗

PubMed2003

The carboxyl terminus of human cytomegalovirus-encoded 7 transmembrane receptor US28 camouflages agonism by mediating constitutive endocytosis.

US28 is one of four 7 transmembrane (7TM) chemokine receptors encoded by human cytomegalovirus and has been shown to both signal and endocytose in a ligand-independent, constitutively active manner. Here we show that the constitutive activity and constitutive endocytosis properties of US28 are separable entities in this viral chemokine receptor. We generated chimeric and mutant US28 proteins that were altered in either their constitutive endocytic (US28 Delta 300, US28 Delta 317, US28-NK1-ctail, and US28-ORF74-ctail) or signaling properties (US28R129A). By using this series of mutants, we show that the cytoplasmic tail domain of US28 per se regulates receptor endocytosis, independent of the signaling ability of the core domain of US28. The constitutive endocytic property of the US28 c-tail was transposable to other 7TM receptors, the herpes virus 8-encoded ORF74 and the tachykinin NK1 receptor (ORF74-US28-ctail and NK1-US28-ctail). Deletion of the US28 C terminus resulted in reduced constitutive endocytosis and consequently enhanced signaling capacity of all receptors tested as assessed by inositol phosphate turnover, NF-kappa B, and cAMP-responsive element-binding protein transcription assays. We further show that the constitutive endocytic property of US28 affects the action of its chemokine ligand fractalkine/CX3CL1 and show that in the absence of the US28 C terminus, fractalkine/CX3CL1 acts as an agonist on US28. This demonstrates for the first time that the endocytic properties of a 7TM receptor can camouflage the agonist properties of a ligand.

Amino Acid Sequence↗

PubMed2003

Changes in hippocampal histamine receptors across the hibernation cycle in ground squirrels.

Hibernation is a physiological state characterized by a dramatic reduction in various functions, such as body temperature, heart rate, and metabolism. The hippocampus is thought to be important for regulation of the hibernation bout because it remains electrophysiologically active throughout this extremely depressed state. The question arises as to what neuronal influences act within the hippocampus during hibernation to sustain its activity. We hypothesized that histaminergic input might be an important contributor. Brain histamine is involved in functions relevant to hibernation, such as the regulation of diurnal rhythms, body temperature, and energy metabolism. Furthermore, we have previously shown that the histaminergic system appears to be activated during the hibernating state. In this study, we used receptor binding autoradiography, in situ hybridization, and GTP-gamma-S binding autoradiography to study changes in histamine receptors across the hibernation bout. We were able to demonstrate an increase in histamine H1 and H2 receptors in the hippocampus during hibernation, whereas the mRNA expression and receptor density of the inhibitory H3 receptor decreased. Histamine H3 receptors were shown to exhibit both histamine-activated and constitutive GTP-gamma-S-binding activity in the ground squirrel hippocampus, both of which decreased during hibernation, indicating a decrease in H3 receptor G-protein activation. Taken together, our results indicate that histamine may be involved in maintaining hibernation by sustaining hippocampal activity, possibly through H1 and H2 receptor activity and decreased inhibition by H3 receptors. The involvement of brain histamine, which is generally thought of as an arousal molecule, in maintaining a depressed state of the brain suggests a more general role for the amine in controlling arousal state.

Afferent Pathways↗

PubMed2002

Identification of the first nonpeptidergic inverse agonist for a constitutively active viral-encoded G protein-coupled receptor.

Human cytomegalovirus (HCMV) encodes a G protein-coupled receptor (GPCR), named US28, which shows homology to chemokine receptors and binds several chemokines with high affinity. US28 induces migration of smooth muscle cells, a feature essential for the development of atherosclerosis, and may serve as a co-receptor for human immunodeficiency virus-type 1 entry into cells. Previously, we have shown that HCMV-encoded US28 displays constitutive activity, whereas its mammalian homologs do not. In this study we have identified a small nonpeptidergic molecule (VUF2274) that inhibits US28-mediated phospholipase C activation in transiently transfected COS-7 cells and in HCMV-infected fibroblasts. Moreover, VUF2274 inhibits US28-mediated HIV entry into cells. In addition, VUF2274 fully displaces radiolabeled RANTES (regulated on activation normal T cell expressed and secreted) binding at US28, apparently with a noncompetitive behavior. Different analogues of VUF2274 have been synthesized and pharmacologically characterized, to understand which features are important for its inverse agonistic activity. Finally, by means of mutational analysis of US28, we have identified a glutamic acid in transmembrane 7 (TM 7), which is highly conserved among chemokine receptors, as a critical residue for VUF2274 binding to US28. The identification of a full inverse agonist provides an important tool to investigate the relevance of US28 constitutive activity in viral pathogenesis.

Animals↗