Evidence that a deviation in the kynurenine pathway aggravates atherosclerotic disease in humans.

BACKGROUND: The metabolism of tryptophan (Trp) along the kynurenine pathway has been shown to carry strong immunoregulatory properties. Several experimental studies indicate that this pathway is a major regulator of vascular inflammation and influences atherogenesis. Knowledge of the role of this pathway in human atherosclerosis remains incomplete. OBJECTIVES: In this study, we performed a multiplatform analysis of tissue samples, in vitro and in vivo functional assays to elucidate the potential role of the kynurenine pathway in human atherosclerosis. METHODS AND RESULTS: Comparison of transcriptomic data from carotid plaques and control arteries revealed an upregulation of enzymes within the quinolinic branch of the kynurenine pathway in the disease state, whilst the branch leading to the formation of kynurenic acid (KynA) was downregulated. Further analyses indicated that local inflammatory responses are closely tied to the deviation of the kynurenine pathway in the vascular wall. Analysis of cerebrovascular symptomatic and asymptomatic carotid stenosis data showed that the downregulation of KynA branch enzymes and reduced KynA production were associated with an increased probability of patients to undergo surgery due to an unstable disease. In vitro, we showed that KynA-mediated signalling through aryl hydrocarbon receptor (AhR) is a major regulator of human macrophage activation. Using a mouse model of peritoneal inflammation, we showed that KynA inhibits leukocyte recruitment. CONCLUSIONS: We have found that a deviation in the kynurenine pathway is associated with an increased probability of developing symptomatic unstable atherosclerotic disease. Our study suggests that KynA-mediated signalling through AhR is an important mechanism involved in the regulation of vascular inflammation.

Glucagon-like peptide 1 (GLP-1).

BACKGROUND: The glucagon-like peptide-1 (GLP-1) is a multifaceted hormone with broad pharmacological potential. Among the numerous metabolic effects of GLP-1 are the glucose-dependent stimulation of insulin secretion, decrease of gastric emptying, inhibition of food intake, increase of natriuresis and diuresis, and modulation of rodent ß-cell proliferation. GLP-1 also has cardio- and neuroprotective effects, decreases inflammation and apoptosis, and has implications for learning and memory, reward behavior, and palatability. Biochemically modified for enhanced potency and sustained action, GLP-1 receptor agonists are successfully in clinical use for the treatment of type-2 diabetes, and several GLP-1-based pharmacotherapies are in clinical evaluation for the treatment of obesity. SCOPE OF REVIEW: In this review, we provide a detailed overview on the multifaceted nature of GLP-1 and its pharmacology and discuss its therapeutic implications on various diseases. MAJOR CONCLUSIONS: Since its discovery, GLP-1 has emerged as a pleiotropic hormone with a myriad of metabolic functions that go well beyond its classical identification as an incretin hormone. The numerous beneficial effects of GLP-1 render this hormone an interesting candidate for the development of pharmacotherapies to treat obesity, diabetes, and neurodegenerative disorders.

Opposite Regulation of Ghrelin and Glucagon-like Peptide-1 by Metabolite G-Protein-Coupled Receptors.

Gut hormones send information about incoming nutrients to the rest of the body and thereby control many aspects of metabolism. The secretion of ghrelin and glucagon-like protein (GLP)-1, two hormones with opposite secretory patterns and opposite actions on multiple targets, is controlled by a limited number of G-protein coupled receptors (GPCRs); half of which recognize and bind dietary nutrient metabolites, metabolites generated by gut microbiota, and metabolites of the host's intermediary metabolism. Most metabolite GPCRs controlling ghrelin secretion are inhibitory, whereas all metabolite receptors controlling GLP-1 secretion are stimulatory. This dichotomy in metabolite sensor function, which is obtained through a combination of differential expression and cell-dependent signaling bias, offers pharmacological targets to stimulate GLP-1 and inhibit ghrelin through the same mechanism.

High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice.

BACKGROUND AND PURPOSE: Human pancreatic polypeptide (hPP) is known to suppress appetite and food intake, thereby representing a potential therapeutic approach against obesity and associated metabolic disorders. The aim of this study was to improve hPP stability by covalent PEGylation with diverse molecular weight polyethylene glycols (PEGs) at two positions using promising lead structures while maintaining target activity. EXPERIMENTAL APPROACH: Modified peptides were synthesized by combined solid-phase and solution-phase peptide synthesis. Their potency was investigated in constitutively expressing human epithelial cells and isolated human colonic mucosa as well as receptor-transfected artificial cell lines. Human blood plasma and porcine liver homogenates were used to examine the in vitro stability of the analogues. The most promising variants were injected s.c. in C57BL/6JRj mice to monitor fasting-induced food intake and bioavailability. KEY RESULTS: In human epithelia and colonic mucosal preparations, activity of the modified hPP peptides depended on the core sequence and latency of the peptides was related to PEG size. Peptides modified with a 22 kDa PEG (PEG22) remained intact in blood plasma and on incubation with liver homogenates for more than 96 h. Finally, hPP2-36 , [K22 (PEG22)]hPP2-36 and [K22 (PEG22),Q34 ]hPP significantly reduced cumulative food intake in mice over 16 h after s.c. administration. CONCLUSIONS AND IMPLICATIONS: Modification with PEG22 at position 22 stabilizes hPP significantly while extending its biological activities and could be used in drug development prospectively.

Oral 2-oleyl glyceryl ether improves glucose tolerance in mice through the GPR119 receptor.

The intestinal G protein-coupled receptor GPR119 is a novel metabolic target involving glucagon-like peptide-1 (GLP-1)-derived insulin-regulated glucose homeostasis. Endogenous and diet-derived lipids, including N-acylethanolamines and 2-monoacylglycerols (2-MAG) activate GPR119. The purpose of this work is to evaluate whether 2-oleoyl glycerol (2-OG) improves glucose tolerance through GPR119, using wild type (WT) and GPR 119 knock out (KO) mice. We here show that GPR119 is essential for 2-OG-mediated release of GLP-1 and CCK from GLUTag cells, since a GPR119 specific antagonist completely abolished the hormone release. Similarly, in isolated primary colonic crypt cultures from WT mice, GPR119 was required for 2-OG-stimulated GLP-1 release while there was no response in crypts from KO mice. In vivo, gavage with 2-oleyl glyceryl ether ((2-OG ether), a stable 2-OG analog with a potency of 5.3 µM for GPR119 with respect to cAMP formation as compared to 2.3 µM for 2-OG), significantly (P < 0.05) improved glucose clearance in WT littermates, but not in GPR119 KO mice. Finally, deletion of GPR119 in mice resulted in lower glucagon levels, whereas the levels of insulin and GIP were unchanged. In the present study we show that 2-OG stimulates GLP-1 secretion through GPR119 activation in vitro, and that fat-derived 2-MAGs are potent candidates for mediating fat-induced GLP-1 release through GPR119 in vivo. © 2016 BioFactors, 42(6):665-673, 2016.

Ghrelin.

BACKGROUND: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism. SCOPE OF REVIEW: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery. MAJOR CONCLUSIONS: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

Expression of the short chain fatty acid receptor GPR41/FFAR3 in autonomic and somatic sensory ganglia.

G-protein-coupled receptor 41 (GPR41) also called free fatty acid receptor 3 (FFAR3) is a Gαi-coupled receptor activated by short-chain fatty acids (SCFAs) mainly produced from dietary complex carbohydrate fibers in the large intestine as products of fermentation by microbiota. FFAR3 is expressed in enteroendocrine cells, but has recently also been shown to be present in sympathetic neurons of the superior cervical ganglion. The aim of this study was to investigate whether the FFAR3 is present in other autonomic and sensory ganglia possibly influencing gut physiology. Cryostat sections were cut of autonomic and sensory ganglia of a transgenic reporter mouse expressing the monomeric red fluorescent protein (mRFP) gene under the control of the FFAR3 promoter. Control for specific expression was also done by immunohistochemistry with an antibody against the reporter protein. mRFP expression was as expected found not only in neurons of the superior cervical ganglion, but also in sympathetic ganglia of the thoracic and lumbar sympathetic trunk. Further, neurons in prevertebral ganglia expressed the mRFP reporter. FFAR3-mRFP-expressing neurons were also present in both autonomic and sensory ganglia such as the vagal ganglion, the spinal dorsal root ganglion and the trigeminal ganglion. No expression was observed in the brain or spinal cord. By use of radioactive-labeled antisense DNA probes, mRNA encoding the FFAR3 was found to be present in cells of the same ganglia. Further, the expression of the FFAR3 in the ganglia of the transgenic mice was confirmed by immunohistochemistry using an antibody directed against the receptor protein, and double labeling colocalized mRFP and the FFAR3-protein in the same neurons. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) on extracts from the ganglia supported the presence mRNA encoding the FFAR3 in most of the investigated tissues. These data indicate that FFAR3 is expressed on postganglionic sympathetic and sensory neurons in both the autonomic and somatic peripheral nervous system and that SCFAs act not only through the enteroendocrine system but also directly by modifying physiological reflexes integrating the peripheral nervous system and the gastro-intestinal tract.

Structural basis for constitutive activity and agonist-induced activation of the enteroendocrine fat sensor GPR119.

BACKGROUND AND PURPOSE: GPR119 is a Gαs-coupled 7TM receptor activated by endogenous lipids such as oleoylethanolamide (OEA) and by the dietary triglyceride metabolite 2-monoacylglycerol. GPR119 stimulates enteroendocrine hormone and insulin secretion. But despite massive drug discovery efforts in the field, very little is known about the basic molecular pharmacology of GPR119. EXPERIMENTAL APPROACH: GPR119 receptor signalling was studied in transfected cells. Mutational mapping (30 mutations in 23 positions) was performed on residues required for ligand-independent and agonist-induced GPR119 activation (AR231453 and OEA). Novel Rosetta-based receptor modelling was applied, using a composite template approach with segments from different X-ray structures and fully flexible ligand docking. KEY RESULTS: The increased signalling induced by increasing the cell surface expression of GPR119 in the absence of agonist and the inhibitory effect of two synthetic inverse agonists demonstrated that GRP119 signals with a high degree of constitutive activity through the Gαs pathway. The mutational maps for AR231453 and OEA were very similar and, surprisingly, also similar to the mutational map for residues affecting the constitutive signalling - albeit with key differences. Surprisingly, almost all residues in extracellular loop-2b were important for the constitutive activity. The molecular modelling and docking demonstrated that AR231453 binds in a 'vertical' pocket in between mutational hits reaching from the centre of the receptor out to extracellular loop-2b. CONCLUSIONS AND IMPLICATIONS: The high constitutive activity of GPR119 should be taken into account in future drug discovery efforts, which can now be guided by the detailed knowledge of the physiochemical properties of the extended ligand-binding pocket.

Glucose metabolism is altered after loss of L cells and α-cells but not influenced by loss of K cells.

The enteroendocrine K and L cells are responsible for secretion of glucose-dependent insulinotropic polypeptide (GIP) and glucagon like-peptide 1 (GLP-1), whereas pancreatic α-cells are responsible for secretion of glucagon. In rodents and humans, dysregulation of the secretion of GIP, GLP-1, and glucagon is associated with impaired regulation of metabolism. This study evaluates the consequences of acute removal of Gip- or Gcg-expressing cells on glucose metabolism. Generation of the two diphtheria toxin receptor cellular knockout mice, TgN(GIP.DTR) and TgN(GCG.DTR), allowed us to study effects of acute ablation of K and L cells and α-cells. Diphtheria toxin administration reduced the expression of Gip and content of GIP in the proximal jejunum in TgN(GIP.DTR) and expression of Gcg and content of proglucagon-derived peptides in both proximal jejunum and terminal ileum as well as content of glucagon in pancreas in TgN(GCG.DTR) compared with wild-type mice. GIP response to oral glucose was attenuated following K cell loss, but oral and intraperitoneal glucose tolerances were unaffected. Intraperitoneal glucose tolerance was impaired following combined L cell and α-cell loss and normal following α-cell loss. Oral glucose tolerance was improved following L cell and α-cell loss and supernormal following α-cell loss. We present two mouse models that allow studies of the effects of K cell or L cell and α-cell loss as well as isolated α-cell loss. Our findings show that intraperitoneal glucose tolerance is dependent on an intact L cell mass and underscore the diabetogenic effects of α-cell signaling. Furthermore, the results suggest that K cells are less involved in acute regulation of mouse glucose metabolism than L cells and α-cells.

Molecular basis for agonism in the BB3 receptor: an epitope located on the interface of transmembrane-III, -VI, and -VII.

Epitopes determining the agonist property of two structurally distinct selective ligands for the human bombesin receptor subtype 3 (BB3), [D-Tyr6,(R)-Apa11,Phe13, Nle14]-bombesin(6-14) (Pep-1) and Ac-Phe-Trp-Ala-His(TauBzl)-Nip-Gly-Arg-NH2 (Pep-2), were mapped through systematic mutagenesis of the main ligand-binding pocket of the receptor. The mutational map for the smaller Pep-2 spanned the entire binding pocket of the BB3 receptor. In contrast, the much fewer mutational hits for the larger Pep-1 were confined to the center of the pocket, i.e., the opposing faces of the extracellular segments of transmembrane (TM)-III, TM-VI, and TM-VII. All the residues, which upon mutation affected Pep-1, were also hits for Pep-2 and included those that were most essential for the function of Pep-2: LeuIII:04 (Leu(123)), TyrVI:16 (Tyr(291)), and ArgVII:06 (Arg(316)). The BB3 receptor was found to signal with 12% ligand-independent activity that was strongly influenced both positively and negatively by several mutations in the binding pocket. The substitutions, which decreased the constitutive signaling, included not only the major mutational hits for the peptide agonists but also mutations more superficially located in the receptor. It is concluded that activation of the BB3 receptor is dependent upon an epitope in the main ligand-binding pocket at the interface between TM-III, TM-VI, and TM-VII that corresponds to the site where, for example, activating metal ion sites have been constructed previously in 7TM receptors.

Targeting CXCR4 in HIV cell-entry inhibition.

CXCR4 and CCR5 constitute the two major coreceptors for HIV-1 entry into host cells. In the course of an HIV-infection, a coreceptor switch takes place in approximately half of the patients - from R5 HIV-1 (CCR5 utilizing) strains to X4 HIV-1 (CXCR4 utilizing) strains. Treatment of HIV-infected individuals with CXCR4 antagonists delays the onset of AIDS by preventing the CCR5 to CXCR4 coreceptor switch. In addition to the endogenous CXCR4 and CCR5 ligands, other chemokines, for example the human herpesvirus 8 encoded CC-chemokine, vCCL2, and modifications hereof, have proven efficient HIV-1 cell-entry inhibition through chemokine receptor interaction. However, pharmacokinetic and immunogenic drawbacks of chemokines and peptidic/peptoid compounds have brought the attention towards small-molecule antagonists, such as AMD3100, that displays high specificity and affinity towards CXCR4, but unfortunately no oral bioavailability. The hunt for orally active small-molecule CXCR4 antagonists led to the development of monocyclam-based compounds, and recently to the non-cyclam antagonist AMD070, which is orally active and currently in Phase II clinical trial as anti-HIV treatment. Current review provides an overview of the drug discovery within the field of anti-HIV treatment targeting CXCR4 spanning from natural occurring and modified chemokines, to HIV-mimicking peptides and peptoids ending at the non-peptide antagonists.

Studies of relationships between variation of the human G protein-coupled receptor 40 Gene and Type 2 diabetes and insulin release.

AIMS: Recently, a novel human G protein-coupled receptor 40 (GPR40), which is predominantly expressed in pancreatic islets, was shown to mediate an amplifying effect of long-chain fatty acids on glucose-induced insulin secretion. The present aim was to examine the coding region of GPR40 for variation and to assess whether identified variants confer an increased risk of Type 2 diabetes or altered insulin release. METHODS: Mutation analysis was performed in 43 patients with Type 2 diabetes, 18 normal glucose-tolerant subjects, and 3 maturity-onset of diabetes in the young (MODY) X patients using direct sequencing. Genotyping was performed using polymerase chain reaction (PCR)-generated primer extension products analysis by high throughput chip-based mass spectrometry (MALDI-TOF). The potential impact of GPR40 mutations on [(3)H]-myo-inositol turnover was estimated in COS-7 cells after stimulation with various concentrations of 5,8,11-eicosatriynoic acid. RESULTS: Two nucleotide substitutions, an Arg211His polymorphism and a rare Asp175Asn mutation, were identified. Both variants showed EC(50) values similar to the wild type. However, the maximal efficacy of the rare Asp175Asn was 39% lower compared with the wild type (P = 0.01). The Arg211His polymorphism had a similar allele frequency among 1384 Type 2 diabetic patients [MAF%; 23.4 (95% CI: 21.8-25.0)] and 4424 middle-aged glucose-tolerant subjects [24.1% (23.2-25.0)]. A genotype-quantitative trait study of 5597 non-diabetic, middle-aged subjects from the Inter99 cohort showed no significant differences in oral glucose tolerance test (OGTT)-derived estimates of insulin release between carriers of various GPR40 genotypes. CONCLUSIONS: Variations in the coding region of GPR40 do not appear to be associated with Type 2 diabetes or insulin release alterations.

Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor.

We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-I receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput Screening (MTS). Using the NovoStar we have found the molecular pharmacology of the NK1 receptor to be comparable to that observed in classical signal transduction assays. Thus, we have observed an EC50 value of 3 nM for substance P induced Ca2+ response. This value corresponds well with previously published values for substance P induced IP and cAMP turnover. [1] Using the NovoStar technology we have studied the pharmacological profile of the well known non-peptide NKI receptor antagonists CP96,345 and SR140,333 [2,3] in respect of inhibition of the Ca2+ response induced by substance P. Interestingly, the antagonistic potency of the antagonists depended greatly on the experimental design, e.g., a dependency of timing in the addition of antagonists vs. agonist was noted. Also, metal-ion site engineered NK1 receptors [2] were tested for the ability of metal-ions to inhibit signalling. It is concluded that the NovoStar is a reliable tool for the study of receptor Ca2+ signalling, both as a research tool and as a MTS system.

Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity.

ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does not respond to agonist stimulation have a much lower incidence of angiogenic lesions and tumors. These results indicate that induction of the KS-like disease in transgenic mice by ORF74 requires not only high constitutive signaling activity but also modulation of this activity by endogenous chemokines.

The first PP supper--Camelot at Bispebjerg.

In the early seventies of the last century, in a temporary building called "Sukkertoppen" at Bispebjerg Hospital, a group of devoted, young medical doctors and a couple of students together with a team of highly skilled laboratory technicians gathered around the "senior" Jens F. Rehfeld, who had barely turned thirty years of age. Each person got his own peptide, raised antibodies, iodinated the peptide and established a radioimmunoassay with which he could then conquer the world of gastrointestinal hormones. It was a unique time, where old hormones had just been chemically characterized and could now be subjected to immunochemical measurements and characterization for the first time. It was also a period when a whole host of new biologically active peptides were being discovered, and basically anything you did with "your" peptide was exciting news for the scientific community. The concept of neuropeptides was being established and Jens Frederik and his brave scientific knights were in the middle of it all. The period ended in the summer of 1976, when Rehfeld moved his court to the University of Aarhus and the others scattered--each to establish their own scientific group.

MC148 encoded by human molluscum contagiosum poxvirus is an antagonist for human but not murine CCR8.

The viral CC chemokines MC148, encoded by the poxvirus molluscum contagiosum, and viral macrophage inflammatory protein (vMIP)-I and vMIP-II, encoded by human herpesvirus 8, were probed on the murine CC receptor (CCR) 8 in parallel with human CCR8. In calcium mobilization assays, vMIP-I acted as a high-affinity agonist, whereas vMIP-II acted as a low-affinity antagonist on the murine CCR8 as well as the human CCR8. MC148 was found to bind and block responses through the human CCR8 with high affinity, but surprisingly MC148 was unable to bind and block responses through the murine CCR8. Because MC148 is the only high-affinity antagonist known to target and be selective for CCR8, MC148 is a valuable tool to decipher the role played by CCR8 in the immune system. This study shows that MC148 could not be used in murine inflammatory models; however, it will be interesting to see whether it can be used in other animal models to delineate the role played by CCR8.

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.

The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling.

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX(3)C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

Mutation of Asp(171) and Asp(262) of the chemokine receptor CXCR4 impairs its coreceptor function for human immunodeficiency virus-1 entry and abrogates the antagonistic activity of AMD3100.

The bicyclam AMD3100 is a highly potent and selective CXCR4 antagonist with strong antiviral activity against human immunodeficiency virus (HIV)-1 and HIV-2, which use CXCR4 as coreceptor for host cell entry. Here, we investigated the interaction of AMD3100 with CXCR4 at the molecular level by mutational analysis. We established a set of stably transfected U87.CD4 cell lines expressing different mutant forms of CXCR4 (i.e., CXCR4[WT], CXCR4[D171N], CXCR4[D262N], CXCR4[D171N,D262N], and CXCR4[H281A]), to compare the activity of the compound against mutated versus wild-type CXCR4. We found that the antagonistic action of AMD3100 against CXCR4--as assessed by the inhibitory effects of the compound on stromal cell-derived factor (SDF-1) binding to its receptor and on SDF-1-induced intracellular calcium signaling, and by displacement of the CXCR4-specific antibody, clone 12G5--was greatly reduced by substitution of Asp(171) and/or Asp(262) by neutral asparagine residue(s). Both aspartates, but most particularly Asp(262), also proved essential for the anti-HIV-1 activity of AMD3100 against the viruses NL4.3, IIIB, and HE. In contrast, substitution of His(281) by a neutral alanine potentiated the antagonistic and antiviral effects of the compound in the different assay systems. Importantly, compared with the wild-type receptor, CXCR4[D262N] was much less effective, whereas CXCR4[D171N,D262N] completely failed as a coreceptor for infection by HIV-1 NL4.3. Thus, the negatively charged aspartate residues at positions 171 and 262, located in transmembrane domains 4 and 6 of the 7-transmembrane receptor, respectively, may represent crucial sites for electrostatic interaction of the positive charges of the bicyclams, as well as for the highly basic V3 loop of the gp120 envelope protein of certain HIV-1 strains.

Virally encoded 7TM receptors.

A number of herpes- and poxviruses encode 7TM G-protein coupled receptors most of which clearly are derived from their host chemokine system as well as induce high expression of certain 7TM receptors in the infected cells. The receptors appear to be exploited by the virus for either immune evasion, cellular reprogramming, tissue targeting or for cell entry. Through their efficient evolutionary machinery and through in vivo selection performed directly on the human cellular and molecular targets, virus have been able to optimize the encoded receptors for distinct pharmacological profiles to help in various parts of the viral life cyclus. Most of the receptors encoded by human pathogenic virus are still orphan receptors, i.e. the endogenous ligand is unknown. In the few cases where it has been possible to characterize these receptors pharmacologically, they have been found to bind a broad spectrum of either CC chemokines, US28 from human cytomegalovirus, or CXC chemokines, ORF74 from human herpesvirus 8. Nevertheless, US28 has been specifically optimized for recognition of the membrane bound chemokine, fractalkine, conceivably involved in cell-cell transfer of virus; whereas ORF74 among the endogenous CXC chemokines has selected angiogenic chemokines as agonists and angiostatic/modulatory chemokines as inverse agonists. ORF74 possess substantial cell-transforming properties and signals with high constitutive activity through the phospholipase C and MAP kinase pathways. Interestingly, transgenic expression of this single gene in certain lymphocyte cell lineages leads to the development of lesions which are remarkably similar to Kaposi's sarcoma, a human herpesvirus 8 associated disease. Thus, this and other virally encoded 7TM receptors appear to be attractive future drug targets.