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Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates. When compared to the native NMU-8 peptide, compounds 16, 18 and 20 have potent agonist activity and affinity for both NMU receptors. Selectivity towards NMUR1 was observed when the Phe residue in position 4 was modified, whereas higher potencies at NMUR2 were found when substitutions of the Pro residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting in half-lifes from 4 min for NMU-8, up to more than 23 h for compound 42., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Background/objective: After digestion, dietary triacylglycerol stimulates incretin release in humans, mainly through generation of 2-monoacylglycerol, an agonist for the intestinal G protein-coupled receptor 119 (GPR119). Enhanced incretin release may have beneficial metabolic effects. However, dietary fat may promote weight gain and should therefore be restricted in obesity. We designed C4-dietary oil (1,3-di-butyryl-2-oleoyl glycerol) as a 2-oleoyl glycerol (2-OG)-generating fat type, which would stimulate incretin release to the same extent while providing less calories than equimolar amounts of common triglycerides, e.g., olive oil., Subjects and Methods: We studied the effect over 180 min of (a) 19 g olive oil plus 200 g carrot, (b) 10.7 g C4 dietary oil plus 200 g carrot and (c) 200 g carrot, respectively, on plasma responses of gut and pancreatic hormones in 13 overweight patients with type 2 diabetes (T2D). Theoretically, both oil meals result in formation of 7.7 g 2-OG during digestion., Results: Both olive oil and C4-dietary oil resulted in greater postprandial (P ≤ 0.01) glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) responses (incremental area under curve (iAUC)): iAUC GLP-1 : 645 ± 194 and 702 ± 97 pM × min; iAUC GIP : 4,338 ± 764 and 2,894 ± 601 pM × min) compared to the carrot meal (iAUC GLP-1 : 7 ± 103 pM × min; iAUC GIP : 266 ± 234 pM × min). iAUC for GLP-1 and GIP were similar for C4-dietary oil and olive oil, although olive oil resulted in a higher peak value for GIP than C4-dietary oil., Conclusion: C4-dietary oil enhanced secretion of GLP-1 and GIP to almost the same extent as olive oil, in spite of liberation of both 2-OG and oleic acid, which also may stimulate incretin secretion, from olive oil. Thus, C4-dietary oil is more effective as incretin releaser than olive oil per unit of energy and may be useful for dietary intervention.

Melatonin has attenuated myocardial ischemia reperfusion injury in experimental studies. We hypothesized that the administration of melatonin during acute myocardial reperfusion improves myocardial salvage index in patients with ST-elevation myocardial infarction. Patients (n = 48) were randomized in a 1:1 ratio to intracoronary and intravenous melatonin (total 50 mg) or placebo. The myocardial salvage index assessed by cardiac magnetic resonance imaging at day 4 (± 1 day) after primary percutaneous coronary intervention was similar in the melatonin group (n = 22) at 55.3% (95% CI 47.0-63.6) and the placebo group (n = 19) at 61.5% (95% CI 57.5-65.5), p = 0.21. The levels of high-sensitive troponin T, creatinine kinase myocardial band, and oxidative biomarkers (advanced oxidation protein products, malondialdehyde, myeloperoxidase) were similar in the groups. The frequency of clinical events at 90 days did not differ between the groups. In conclusion, melatonin did not improve the myocardial salvage index after primary percutaneous coronary intervention in patients with ST elevation myocardial infarction compared with placebo.

Psoriasis is a chronic inflammatory skin disease characterized by the infiltration of T cell and other immune cells to the skin in response to injury or autoantigens. Conventional, as well as unconventional, γδ T cells are recruited to the dermis and epidermis by CCL20 and other chemokines. Together with its receptor CCR6, CCL20 plays a critical role in the development of psoriasiform dermatitis in mouse models. We screened a panel of CCL20 variants designed to form dimers stabilized by intermolecular disulfide bonds. A single-atom substitution yielded a CCL20 variant (CCL20 S64C) that acted as a partial agonist for the chemokine receptor CCR6. CCL20 S64C bound CCR6 and induced intracellular calcium release, consistent with G-protein activation, but exhibited minimal chemotactic activity. Instead, CCL20 S64C inhibited CCR6-mediated T cell migration with nominal impact on other chemokine receptor signaling. When given in an IL-23-dependent mouse model for psoriasis, CCL20 S64C prevented psoriatic inflammation and the up-regulation of IL-17A and IL-22. Our results validate CCR6 as a tractable therapeutic target for psoriasis and demonstrate the value of CCL20 S64C as a lead compound., Competing Interests: Conflict of interest statement: B.F.V. and F.C.P. have ownership interests in Protein Foundry, LLC. The other authors state no conflict of interest. The technology presented is protected under provisional patent filing no. 62/170,347.

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in β cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

A truncated form of human glucose-dependent insulinotropic polypeptide (GIP), GIP(3-30)NH 2 , was recently identified as an antagonist of the human GIP receptor. This study examined the ability of GIP(3-30)NH 2 to antagonize the physiological actions of GIP in glucose metabolism, subcutaneous abdominal adipose tissue blood flow (ATBF), and lipid metabolism in humans. Eight lean subjects were studied by measuring arteriovenous concentrations of metabolites and ATBF on three different occasions during hyperglycemic-hyperinsulinemic clamps with concomitant infusions of GIP, GIP(3-30)NH 2 , or both GIP and GIP(3-30)NH 2 During infusion of GIP(3-30)NH 2 alone and in combination with GIP, insulin levels and the total glucose amount infused to maintain the clamp were lower than during GIP alone. In addition, ATBF remained constant during the antagonist and increased only slightly in combination with GIP, whereas it increased fivefold during GIP alone. Adipose tissue triacylglyceride (TAG) and glucose uptake decreased, and the free fatty acid/glycerol ratio increased during the antagonist alone and in combination with GIP. The changes in glucose infusion rates and plasma insulin levels demonstrate an inhibitory effect of the antagonist on the incretin effect of GIP. In addition, the antagonist inhibited GIP-induced increase in ATBF and decreased the adipose tissue TAG uptake, indicating that GIP also plays a crucial role in lipid metabolism., (© 2017 by the American Diabetes Association.)

Background and Purpose: The GPCR Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored., Experimental Approach: We investigated the effects of the EBI2 endogenous agonist 7α,25-dihydroxycholesterol (7α,25-OHC) on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of the EBI2 receptor to investigate the structural basis of the allosteric modulation by sodium., Key Results: Residue N114, located in the middle of transmembrane-III at position III:11/3.35, was found to function as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolished heterotrimeric G protein subunit Gi α activation by 7α,25-OHC even though the specific binding of [ 3 H]-7α,25-OHC increased. In contrast, the N114A mutant was still able to recruit β-arrestin and even had an enhanced potency (18.7-fold) compared with EBI2 wild type. Sodium had a negative allosteric effect on oxysterol binding that was mediated via N114, verifying the key role of N114. This was further supported by molecular modelling of the ion binding site based on a EBI2 receptor homology model., Conclusions and Implications: Collectively, our data point to N114 as a key residue for EBI2 signalling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding., (© 2017 The British Pharmacological Society.)

Varicella zoster virus (VZV) is a highly prevalent human pathogen that establishes latency in neurons of the peripheral nervous system. Primary infection causes varicella whereas reactivation results in zoster, which is often followed by chronic pain in adults. Following infection of epithelial cells in the respiratory tract, VZV spreads within the host by hijacking leukocytes, including T cells, in the tonsils and other regional lymph nodes, and modifying their activity. In spite of its importance in pathogenesis, the mechanism of dissemination remains poorly understood. Here we addressed the influence of VZV on leukocyte migration and found that the purified recombinant soluble ectodomain of VZV glycoprotein C (rSgC) binds chemokines with high affinity. Functional experiments show that VZV rSgC potentiates chemokine activity, enhancing the migration of monocyte and T cell lines and, most importantly, human tonsillar leukocytes at low chemokine concentrations. Binding and potentiation of chemokine activity occurs through the C-terminal part of gC ectodomain, containing predicted immunoglobulin-like domains. The mechanism of action of VZV rSgC requires interaction with the chemokine and signalling through the chemokine receptor. Finally, we show that VZV viral particles enhance chemokine-dependent T cell migration and that gC is partially required for this activity. We propose that VZV gC activity facilitates the recruitment and subsequent infection of leukocytes and thereby enhances VZV systemic dissemination in humans.

The chemokine CXCL12 or stromal cell-derived factor 1/SDF-1 attracts hematopoietic progenitor cells and mature leukocytes through the G protein-coupled CXC chemokine receptor 4 (CXCR4). In addition, it interacts with atypical chemokine receptor 3 (ACKR3 or CXCR7) and glycosaminoglycans. CXCL12 activity is regulated through posttranslational cleavage by CD26/dipeptidyl peptidase 4 that removes two NH 2 -terminal amino acids. CD26-truncated CXCL12 does not induce calcium signaling or chemotaxis of mononuclear cells. CXCL12(3-68) was chemically synthesized de novo for detailed biological characterization. Compared to unmodified CXCL12, CXCL12(3-68) was no longer able to signal through CXCR4 via inositol trisphosphate (IP 3 ), Akt or extracellular signal-regulated kinases 1 and 2 (ERK1/2). Interestingly, the recruitment of β-arrestin 2 to the cell membrane via CXCR4 by CXCL12(3-68) was abolished, whereas a weakened but significant β-arrestin recruitment remained via ACKR3. CXCL12-induced endothelial cell migration and signal transduction was completely abrogated by CD26. Intact CXCL12 hardly induced lymphocyte migration upon intra-articular injection in mice. In contrast, oral treatment of mice with the CD26 inhibitor sitagliptin reduced CD26 activity and CXCL12 cleavage in blood plasma. The potential of CXCL12 to induce intra-articular lymphocyte infiltration was significantly increased in sitagliptin-treated mice and CXCL12(3-68) failed to induce migration under both CD26-inhibiting and non-inhibiting conditions. In conclusion, CD26-cleavage skews CXCL12 towards β-arrestin dependent recruitment through ACKR3 and destroys the CXCR4-mediated lymphocyte chemoattractant properties of CXCL12 in vivo. Hence, pharmacological CD26-blockade in tissues may enhance CXCL12-induced inflammation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Alternative processing of the precursor protein pro-GIP results in endogenously produced GIP(1-30)NH 2 , that by DPP-4 cleavage in vivo results in the metabolite GIP(3-30)NH 2 . We showed previously that GIP(3-30)NH 2 is a high affinity antagonist of the human GIPR in vitro. Here we determine whether it is suitable for studies of GIP physiology in rats since effects of GIP agonists and antagonists are strictly species-dependent. Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation or assayed in competition binding using human 125 I-GIP(1-42) as radioligand. In isolated perfused rat pancreata, insulin, glucagon, and somatostatin-releasing properties were evaluated. Competition binding demonstrated that on the rat GIP receptor (GIPR), rat GIP(3-30)NH 2 bound with high affinity (K i of 17nM), in contrast to human GIP(3-30)NH 2 (K i of 250nM). In cAMP studies, rat GIP(3-30)NH 2 inhibited GIP(1-42)-induced rat GIPR activation and schild-plot analysis showed competitive antagonism with a pA 2 of 13nM and a slope of 0.9±0.09. Alone, rat GIP(3-30)NH 2 displayed weak, low-potent partial agonistic properties (EC 50 >1μM) with an efficacy of 9.4% at 0.32μM compared to GIP(1-42). In perfused rat pancreata, rat GIP(3-30)NH 2 efficiently antagonized rat GIP(1-42)-induced insulin, somatostatin, and glucagon secretion. In summary, rat GIP(3-30)NH 2 is a high affinity competitive GIPR antagonist and effectively antagonizes GIP-mediated G protein-signaling as well as pancreatic hormone release, while human GIP(3-30)NH 2 , despite a difference of only one amino acid between the two (arginine in position 18 in rat GIP(3-30)NH 2 ; histidine in human), is unsuitable in the rat system. This underlines the importance of species differences in the GIP system, and the limitations of testing human peptides in rodent systems., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glucose stimulates the secretion of the incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is debated whether the sweet taste receptor (STR) triggers this secretion. We investigated the role of STR activation for glucose-stimulated incretin secretion from an isolated perfused rat small intestine and whether selective STR activation by artificial sweeteners stimulates secretion. Intra-luminal administration of the STR agonists, acesulfame K (3.85% w/v ), but not sucralose (1.25% w/v ) and stevioside (2.5% w/v ), stimulated GLP-1 secretion (acesulfame K: 31 ± 3 pmol/L vs. 21 ± 2 pmol/L, p < 0.05, n = 6). In contrast, intra-arterial administration of sucralose (10 mM) and stevioside (10 mM), but not acesulfame K, stimulated GLP-1 secretion (sucralose: 51 ± 6 pmol/L vs. 34 ± 4 pmol/L, p < 0.05; stevioside: 54 ± 6 pmol/L vs. 32 ± 2 pmol/L, p < 0.05, n = 6), while 0.1 mM and 1 mM sucralose did not affect the secretion. Luminal glucose (20% w/v ) doubled GLP-1 and GIP secretion, but basolateral STR inhibition by gurmarin (2.5 µg/mL) or the inhibition of the transient receptor potential cation channel 5 (TRPM5) by triphenylphosphine oxide (TPPO) (100 µM) did not attenuate the responses. In conclusion, STR activation does not drive GIP/GLP-1 secretion itself, nor does it have a role for glucose-stimulated GLP-1 or GIP secretion.

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5 + B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 ( EBI2 or GPR183 ) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5 + B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.

Reactivation of human cytomegalovirus (HCMV) in transplant recipients can cause life-threatening disease. Consequently, for transplant recipients, killing latently infected cells could have far-reaching clinical benefits. In vivo, myeloid cells and their progenitors are an important site of HCMV latency, and one viral gene expressed by latently infected myeloid cells is US28. This viral gene encodes a cell surface G protein-coupled receptor (GPCR) that binds chemokines, triggering its endocytosis. We show that the expression of US28 on the surface of latently infected cells allows monocytes and their progenitor CD34+ cells to be targeted and killed by F49A-FTP, a highly specific fusion toxin protein that binds this viral GPCR. As expected, this specific targeting of latently infected cells by F49A-FTP also robustly reduces virus reactivation in vitro. Consequently, such specific fusion toxin proteins could form the basis of a therapeutic strategy for eliminating latently infected cells before haematopoietic stem cell transplantation., Competing Interests: The authors declare no competing financial interests.

Here we report a series of close analogues of our recently published scaffold-based tripeptidomimetic CXCR4 antagonists, containing positively charged guanidino groups in R 1 and R 2 , and an aromatic group in R 3 . While contraction/elongation of the guanidine carrying side chains (R 1 and R 2 ) resulted in loss of activity, introduction of bromine in position 1 on the naphth-2-ylmethyl moiety (R 3 ) resulted in an EC 50 of 61μM (mixture of diastereoisomers) against wild-type CXCR4; thus, the antagonistic activity of these tripeptidomimetics seems to be amenable to optimization of the aromatic moiety. Moreover, for analogues carrying a naphth-2-ylmethyl substituent, we observed that a Pictet-Spengler like cyclization side reaction depended on the nature of the R 1 substituent., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX 3 CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX 3 CR1, the endogenous receptor for CX 3 CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus may be targeted by FTPs., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

G protein-coupled receptors (GPCRs) constitute a large protein family of seven transmembrane (7TM) spanning proteins that regulate multiple physiological functions. GPR87 is overexpressed in several cancers and plays a role in tumor cell survival. Here, the basal activity of GPR87 was investigated in transiently transfected HEK293 cells, revealing ligand-independent coupling to Gα i , Gα q and Gα 12/13 . Furthermore, GPR87 showed a ligand-independent G protein-dependent activation of the downstream transcription factors CREB, NFκB, NFAT and SRE. In tetracycline-induced Flp-In T-Rex-293 cells, GPR87 induced cell clustering presumably through Gα 12/13 coupling. In a foci formation assay using retrovirally transduced NIH3T3 cells, GPR87 showed a strong in vitro transforming potential, which correlated to the in vivo tumor induction in nude mice. Importantly, we demonstrate that the transforming potential of GPR87 was correlated to the receptor signaling, as the signaling-impaired mutant R139A (Arg in the conserved "DRY"-motif at the bottom of transmembrane helix 3 of GPR87 substituted to Ala) showed a lower in vitro cell transformation potential. Furthermore, R139A lost the ability to induce cell clustering. In summary, we show that GPR87 is active through several signaling pathways and that the signaling activity is linked to the receptor-induced cell transformation and clustering. The robust surface expression of GPR87 and general high druggability of GPCRs make GPR87 an attractive future anticancer target for drugs that - through inhibition of the receptor signaling - will inhibit its transforming properties., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn 2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn 2+ is anchored to the chemokine receptor-conserved Glu-283 VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248 VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116 III:16/3.40 , a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125 I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37 I:07/1.39 , Trp-86 II:20/2.60 , and Phe-109 III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Some herpesviruses like human cytomegalovirus (HCMV) encode viral G protein-coupled receptors that cause reprogramming of cell signaling to facilitate dissemination of the virus, prevent immune surveillance and establish life-long latency. Human GPCRs are known to function in complex signaling networks involving direct physical interactions as well as indirect crosstalk of orthogonal signaling networks. The human chemokine receptor CXCR4 is expressed on hematopoietic stem cells, leukocytes, endothelial and epithelial cells, which are infected by HCMV or display reservoirs of latency., Results: We investigated the potential heteromerization of US28 with CXCR4 as well as the influence of US28 on CXCR4 signaling. Using Bioluminescence Resonance Energy Transfer and luciferase-complementation based methods we show that US28 expression exhibits negative effects on CXCR4 signaling and constitutive surface expression in HEK293T cells. Furthermore, we demonstrate that this effect is not mediated by receptor heteromerization but via signaling crosstalk. Additionally, we show that in HCMV, strain TB40E, infected HUVEC the surface expression of CXCR4 is strongly downregulated, whereas in TB40E-delUS28 infected cells, CXCR4 surface expression is not altered in particular at late time points of infection., Conclusions: We show that the vGPCR US28 is leading to severely disturbed signaling and surface expression of the chemokine receptor CXCR4 thereby representing an effective mechanism used by vGPCRs to reprogram host cell signaling. In contrast to other studies, we demonstrate that these effects are not mediated via heteromerization.

The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

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., (© 2016 International Union of Biochemistry and Molecular Biology.)

GPR119 is a Gαs-coupled lipid-sensor in the gut, where it mediates release of incretin hormones from the enteroendocrine cells and in pancreatic α-cells, where it releases insulin. Naturally occurring lipids such as monoacylglycerols (MAGs) and N-acylethanolamines (NAEs), like oleoylethanolamide (OEA), activate GPR119, and multiple synthetic ligands have been described. Here, we extend the GPR119 signaling profile to Gαq and Gαi in addition to β-arrestin recruitment and the downstream transcription factors CRE (cAMP response element), SRE (serum response element) and NFAT (nuclear factor of activated T cells). The endogenous OEA and the synthetic AR231453 were full agonists in all pathways except for NFAT, where no ligand-modulation was observed. The potency of AR231453 varied <16-fold (EC 50 from 6 to 95nM) across the different signaling pathways, whereas that of OEA varied >175-fold (from 85nM to 15μM) indicating a biased signaling for OEA. The degree of constitutive activity was 1-10%, 10-30% and 30-70% of OEA-induced E max in Gαi, Gαq and Gαs-driven pathways, respectively. This coincided with the lowest and highest OEA potency observed in Gαi and Gαs-driven pathways, respectively. Incubation for 2h with the 2-MAG-lipase inhibitor JZL84 doubled the constitutive activity, indicating that endogenous lipids contribute to the apparent constitutive activity. Finally, besides being an agonist, AR231453 acted as a positive allosteric modulator of OEA and increased its potency by 54-fold at 100nM AR231453. Our studies uncovering broad and biased signaling, masked constitutive activity by endogenous MAGs, and ago-allosteric properties of synthetic ligands may explain why many GPR119 drug-discovery programs have failed so far., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1 fusion inhibitors. A virus-free cell-based fusion reporter assay, based on mixing "effector cells" (expressing HIV Env and luciferase activator) with "target cells" (expressing CD4, CCR5 wild type or a selection of well-described mutations, and luciferase reporter), was used as fusion readout. Receptor expression was evaluated by ELISA and fluorescence microscopy. On CCR5 WT, Maraviroc and Aplaviroc inhibited fusion with high potencies (EC 50 values of 91 and 501 nM, respectively), whereas removal of key residues for both antagonists (Glu283Ala) or Maraviroc alone (Tyr251Ala) prevented fusion inhibition, establishing this assay as suitable for screening of HIV entry inhibitors. Both ligands inhibited HIV fusion on signaling-deficient CCR5 mutations (Tyr244Ala and Trp248Ala). Moreover, the steric hindrance CCR5 mutation (Gly286Phe) impaired fusion, presumably by a direct hindrance of gp120 interaction. Finally, the efficacy switch mutation (Leu203Phe) - converting small-molecule antagonists/inverse agonists to full agonists biased toward G-protein activation - uncovered that also small-molecule agonists can function as direct HIV-1 cell entry inhibitors. Importantly, no agonist-induced receptor internalization was observed for this mutation. Our studies of the pharmacodynamic requirements for HIV-1 fusion inhibitors highlight the possibility of future development of biased ligands with selective targeting of the HIV-CCR5 interaction without interfering with the normal functionality of CCR5.

The chemokine CXCL12/stromal cell-derived factor-1 is important for leukocyte migration to lymphoid organs and inflamed tissues and stimulates tumor development. In vitro, CXCL12 activity through CXCR4 is abolished by proteolytic processing. However, limited information is available on in vivo effects of posttranslationally modified CXCL12. Natural CXCL12 was purified from the coculture supernatant of stromal cells stimulated with leukocytes and inflammatory agents. In this conditioned medium, CXCL12 with a nitration on Tyr7, designated [3-NT7]CXCL12, was discovered via Edman degradation. CXCL12 and [3-NT7]CXCL12 were chemically synthesized to evaluate the biological effects of this modification. [3-NT7]CXCL12 recruited β-arrestin 2 and phosphorylated the Akt kinase similar to CXCL12 in receptor-transfected cells. Also the affinity of CXCL12 and [3-NT7]CXCL12 for glycosaminoglycans, the G protein-coupled chemokine receptor CXCR4 and the atypical chemokine receptor ACKR3 were comparable. However, [3-NT7]CXCL12 showed a reduced ability to enhance intracellular calcium concentrations, to generate inositol triphosphate, to phosphorylate ERK1/2 and to induce monocyte and lymphocyte chemotaxis in vitro. Moreover, nitrated CXCL12 failed to induce in vivo extravasation of lymphocytes to the joint. In summary, nitration on Tyr7 under inflammatory conditions is a novel natural posttranslational regulatory mechanism of CXCL12 which may downregulate the CXCR4-mediated inflammatory and tumor-promoting activities of CXCL12., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article.

Background Aims: Maturation of dendritic cells (DCs) induces their homing from peripheral to lymphatic tissues guided by CCL21. However, in vitro matured human monocyte-derived DC cancer vaccines injected intradermally migrate poorly to lymph nodes (LNs). In vitro maturation protocols generate DCs with high (type 1 DCs) or low (prostaglandin E2 [PGE2]-DCs) autocrine CCL19 levels, which may potentially interfere with LN homing of DCs., Methods: Employing a three-dimensional (3D) chemotaxis assay, chemokine competition/desensitization studies and short interfering RNA (siRNA) against CCL19, we analyzed the effect of autocrine CCL19 on in vitro migration of human DCs toward CCL21., Results: Using human monocyte-derived DCs in a 3D chemotaxis assay, we are the first to demonstrate that CCL19 more potently induces directed migration of human DCs compared with CCL21. When comparing migration of type 1 DCs and PGE2-DCs, migration of type 1 DCs was strikingly impaired compared with PGE2-DCs, but only toward low concentrations of CCL21. When type 1 DCs were cultured overnight in fresh culture medium (reducing autocrine CCL19 levels), a rescuing effect was observed on migration toward low concentrations of CCL21 in a 3D chemotaxis assay. Finally pre-incubation with CCL19 negatively affected PGE2-DC migration, whereas silencing of CCL19 by siRNA improved type 1 DC migration. Importantly, in both cases, the effect was observed only at low concentrations of CCL21., Conclusions: Our results demonstrate that autocrine CCL19 negatively affects DC migratory potential toward CCL21, the potency difference between CCL19 and CCL21 being the underlying cause. CCL19 secretion level of in vitro matured DCs is an important indicator of DC vaccine homing potential., (Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr(184) (Cys + 1) and Tyr(187) (Cys + 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr(187) in TMIV (Phe(171)) and TMV (Trp(194)). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr(187) This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

There is a constant need for new therapeutic interventions in a wide range of infectious diseases. Over the past few years, the immunotoxins have entered the stage as promising antiviral treatments. Immunotoxins have been extensively explored in cancer treatment and have achieved FDA approval in several cases. Indeed, the design of new anticancer immunotoxins is a rapidly developing field. However, at present, several immunotoxins have been developed targeting a variety of different viruses with high specificity and efficacy. Rather than blocking a viral or cellular pathway needed for virus replication and dissemination, immunotoxins exert their effect by killing and eradicating the pool of infected cells. By targeting a virus-encoded target molecule, it is possible to obtain superior selectivity and drastically limit the side effects, which is an immunotoxin-related challenge that has hindered the success of immunotoxins in cancer treatment. Therefore, it seems beneficial to use immunotoxins for the treatment of virus infections. One recent example showed that targeting of virus-encoded 7 transmembrane (7TM) receptors by immunotoxins could be a future strategy for designing ultraspecific antiviral treatment, ensuring efficient internalization and hence efficient eradication of the pool of infected cells, both in vitro and in vivo. In this review, we provide an overview of the mechanisms of action of immunotoxins and highlight the advantages of immunotoxins as future anti-viral therapies., (© Society for Leukocyte Biology.)

Peptide YY (PYY) is a 36-amino-acid peptide released from enteroendocrine cells upon food intake. The NH2 terminally truncated metabolite, PYY3-36, exerts anorexic effects and has received considerable attention as a possible antiobesity drug target. The kinetics and degradation products of PYY metabolism are not well described. A related peptide, neuropeptide Y, may be degraded from the COOH terminus, and in vivo studies in pigs revealed significant COOH-terminal degradation of PYY. We therefore investigated PYY metabolism in vitro after incubation in human blood and plasma and in vivo after infusion of PYY1-36 and PYY3-36 in eight young, healthy men. A metabolite, corresponding to PYY3-34, was formed after incubation in plasma and blood and during the infusion of PYY. PYY3-34 exhibited no agonistic or antagonistic effects on the Y2 receptor. PYY1-36 infused with and without coadministration of sitagliptin was eliminated with half-lives of 10.1 ± 0.5 and 9.4 ± 0.8 min (means ± SE) and metabolic clearance rates of 15.7 ± 1.5 and 14.1 ± 1.1 ml·kg(-1)·min(-1) after infusion, whereas PYY3-36 was eliminated with a significantly longer half-life of 14.9 ± 1.3 min and a metabolic clearance rate of 9.4 ± 0.6 ml·kg(-1)·min(-1) We conclude that, upon intravenous infusion in healthy men, PYY is inactivated by cleavage of the two COOH-terminal amino acids. In healthy men, PYY3-36 has a longer half-life than PYY1-36., (Copyright © 2016 the American Physiological Society.)

In this work we report a design, synthesis, and detailed functional characterization of unique strongly biased allosteric agonists of CXCR3 that contain tetrahydroisoquinoline carboxamide cores. Compound 11 (FAUC1036) is the first strongly biased allosteric agonist of CXCR3 that selectively induces weak chemotaxis and leads to receptor internalization and the β-arrestin 2 recruitment with potency comparable to that of the chemokine CXCL11 without any activation of G proteins. A subtle structural change (addition of a methoxy group, 14 (FAUC1104)) led to a contrasting biased allosteric partial agonist that activated solely G proteins, induced chemotaxis, but failed to induce receptor internalization or β-arrestin 2 recruitment. Concomitant structure-activity relationship studies indicated very steep structure-activity relationships, which steer the ligand bias between the β-arrestin 2 and G protein pathway. Overall, the information presented provides a powerful platform for further development and rational design of strongly biased allosteric agonists of CXCR3.

Background and Purpose: Glucose-dependent insulinotropic polypeptide (GIP) affects lipid, bone and glucose homeostasis. High-affinity ligands for the GIP receptor are needed to elucidate the physiological functions and pharmacological potential of GIP in vivo. GIP(1-30)NH2 is a naturally occurring truncation of GIP(1-42). Here, we have characterized eight N-terminal truncations of human GIP(1-30)NH2 ., Experimental Approach: COS-7 cells were transiently transfected with human GIP receptors and assessed for cAMP accumulation upon ligand stimulation or competition binding with (125) I-labelled GIP(1-42), GIP(1-30)NH2 , GIP(2-30)NH2 or GIP(3-30)NH2 ., Key Results: GIP(1-30)NH2 displaced (125) I-GIP(1-42) as effectively as GIP(1-42) (Ki 0.75 nM), whereas the eight truncations displayed lower affinities (Ki 2.3-347 nM) with highest affinities for GIP(3-30)NH2 and GIP(5-30)NH2 (5-30)NH2 . Only GIP(1-30)NH2 (Emax 100% of GIP(1-42)) and GIP(2-30)NH2 (Emax 20%) were agonists. GIP(2- to 9-30)NH2 displayed antagonism (IC50 12-450 nM) and Schild plot analyses identified GIP(3-30)NH2 and GIP(5-30)NH2 as competitive antagonists (Ki 15 nM). GIP(3-30) NH2 was a 26-fold more potent antagonist than GIP(3-42). Binding studies with agonist ((125) I-GIP(1-30)NH2 ), partial agonist ((125) I-GIP(2-30)NH2 ) and competitive antagonist ((125) I-GIP(3-30)NH2 ) revealed distinct receptor conformations for these three ligand classes., Conclusions and Implications: The N-terminus is crucial for GIP agonist activity. Removal of the C-terminus of the endogenous GIP(3-42) creates another naturally occurring, more potent, antagonist GIP(3-30)NH2 , which like GIP(5-30)NH2 , was a high-affinity competitive antagonist. These peptides may be suitable tools for basic GIP research and future pharmacological interventions., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

This crossover study investigated the pharmacokinetics and adverse effects of high-dose intravenous melatonin. Volunteers participated in 3 identical study sessions, receiving an intravenous bolus of 10 mg melatonin, 100 mg melatonin, and placebo. Blood samples were collected at baseline and 0, 60, 120, 180, 240, 300, 360, and 420 minutes after the bolus. Quantitative determination of plasma melatonin concentrations was performed using a radioimmunoassay technique. Pharmacokinetic parameters were estimated by a compartmental pharmacokinetic analysis. Adverse effects included assessments of sedation and registration of other symptoms. Sedation, evaluated as simple reaction times, was measured at baseline and 120, 180, 300, and 420 minutes after the bolus. Twelve male volunteers completed the study. Median (IQR) Cmax after the bolus injections of 10 mg and 100 mg of melatonin were 221,500.0 (185,637.5-326,175.0) pg/mL and 1,251,500.0 (864,375.0-1,770,500.0) pg/mL, respectively; mean (SD) t1/2 was 42.3 (5.6) minutes and 46.2 (6.2) minutes; mean (SD) Vd was 1.6 (0.9) L/kg and 2.0 (0.8) L/kg; mean (SD) CL was 0.0253 (0.0096) L/min · kg and 0.0300 (0.0120) L/min · kg; and median (IQR) AUC0- ∞ , 8,997,633.0 (6,071,696.2-11,602,811.9) pg · min/mL and 54,685,979.4 (36,028,638.6-105,779,612.0) pg · min/mL. High-dose intravenous melatonin did not induce sedation, evaluated as simple reaction times. No adverse effects were reported in the study., (© 2015, The American College of Clinical Pharmacology.)

Background: The aim was to investigate the pharmacokinetics of oral and iv melatonin in healthy volunteers., Methods: The study was performed as a cohort crossover study. The volunteers received either 10 mg oral melatonin or 10 mg intravenous melatonin on two separate study days. Blood samples were collected at different time points following oral administration and short iv infusion, respectively. Plasma melatonin concentrations were determined by RIA technique. Pharmacokinetic analyses were performed by "the method of residuals" and compartmental analysis. The pharmacokinetic variables: k a, t 1/2 absorption, t max, C max, t 1/2 elimination, AUC 0-∞, and bioavailability were determined for oral melatonin. C max, t 1/2 elimination, V d, CL and AUC 0-∞ were determined for intravenous melatonin., Results: Twelve male volunteers completed the study. Baseline melatonin plasma levels did not differ significantly between the study days (P = 0.067). Mean (SD) t 1/2 absorption of oral melatonin was 6.0 (3.1) min. Mean t max was 40.8 (17.8) min with a median (IQR) C max of 3550.5 (2500.5-8057.5) pg ml(-1). Mean t 1/2 elimination was 53.7 (7.0) min. Median absolute bioavailability was 2.5 (1.7-4.7) %. Median C max after short iv infusion of melatonin was 389,875.0 (174,775.0-440,362.5) pg ml(-1). Mean t 1/2 elimination was 39.4 (3.6) min, mean V d 1.2 (0.6) l kg(-1) and mean CL 0.0218 (0.0102) l min(-1) kg(-1)., Conclusions: This cohort crossover study estimated pharmacokinetics of oral and iv melatonin, respectively in healthy volunteers. Bioavailability of oral melatonin was only 3 %., Trial Registration: Eudra-CT number: 2013-000205-23 (initial registration 27.03.2013). Clinicaltrials.gov Identifier: NCT01923974 (initial registration 08.08.2013).

The chemokine system mediates leukocyte migration during homeostatic and inflammatory processes. Traditionally, it is described as redundant and promiscuous, with a single chemokine ligand binding to different receptors and a single receptor having several ligands. Signaling of chemokine receptors occurs via two major routes, G protein- and β-arrestin-dependent, which can be preferentially modulated depending on the ligands or receptors involved, as well as the cell types or tissues in which the signaling event occurs. The preferential activation of a certain signaling pathway to the detriment of others has been termed signaling bias and can accordingly be grouped into ligand bias, receptor bias, and tissue bias. Bias has so far been broadly overlooked in the process of drug development. The low number of currently approved drugs targeting the chemokine system, as well as the broad range of failed clinical trials, reflects the need for a better understanding of the chemokine system. Thus, understanding the character, direction, and consequence of biased signaling in the chemokine system may aid the development of new therapeutics. This review describes experiments to assess G protein-dependent and -independent signaling in order to quantify chemokine system bias., (© 2016 Elsevier Inc. All rights reserved.)

Background and Purpose: Specific, high potency receptor antagonists are valuable tools when evaluating animal and human physiology. Within the glucose-dependent, insulinotropic polypeptide (GIP) system, considerable attention has been given to the presumed GIP receptor antagonist, (Pro3)GIP, and its effect in murine studies. We conducted a pharmacological analysis of this ligand including interspecies differences between the rodent and human GIP system., Experimental Approach: Transiently transfected COS-7 cells were assessed for cAMP accumulation upon ligand stimulation and assayed in competition binding using (125) I-human GIP. Using isolated perfused pancreata both from wild type and GIP receptor-deficient rodents, insulin-releasing, glucagon-releasing and somatostatin-releasing properties in response to species-specific GIP and (Pro3)GIP analogues were evaluated., Key Results: Human (Pro3)GIP is a full agonist at human GIP receptors with similar efficacy (Emax ) for cAMP production as human GIP, while both rat and mouse(Pro3)GIP were partial agonists on their corresponding receptors. Rodent GIPs are more potent and efficacious at their receptors than human GIP. In perfused pancreata in the presence of 7 mM glucose, both rodent (Pro3)GIP analogues induced modest insulin, glucagon and somatostatin secretion, corresponding to the partial agonist activities observed in cAMP production., Conclusions and Implications: When evaluating new compounds, it is important to consider interspecies differences both at the receptor and ligand level. Thus, in rodent models, human GIP is a comparatively weak partial agonist. Human (Pro3)GIP was not an antagonist at human GIP receptors, so there is still a need for a potent antagonist in order to elucidate the physiology of human GIP., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Treatment with liraglutide leads to weight loss. We investigated whether blood-to-cerebrospinal fluid (CSF) transfer of liraglutide occurs, and if so, whether it associates with clinical weight loss following liraglutide treatment in humans. We performed lumbar puncture and blood sampling in eight patients with type 2 diabetes (mean (range)): age 63 (54-79) years; actual body weight: 90 (75-118) kg treated with 1.8 mg liraglutide for 14 (5-22) months and with a treatment-induced weight loss of 8.4 (7-11) kg. We measured liraglutide in plasma and CSF with a radioimmunoassay specific for the N-terminus of the GLP-1 moiety of liraglutide. Mean plasma liraglutide was 31 (range: 21-63) nmol l(-1). The mean CSF-liraglutide concentration was 6.5 (range: 0.9-13.9) pmol l(-1). Ratio of CSF: plasma-liraglutide concentrations was 0.02 (range: 0.07-0.002)% and plasma liraglutide did not correlate with CSF-liraglutide levels (P=0.67). Body weight loss tended to correlate with plasma-liraglutide levels (P=0.06), but not with CSF-liraglutide levels (P=0.69). In conclusion, we measured very low concentrations of liraglutide in CSF, and the levels of CSF liraglutide did not correlate with the actual clinical weight loss in these patients. The amount of liraglutide in plasma tended to correlate with the clinical weight loss.

We here report an experimentally verified binding mode for the known tripeptidomimetic CXCR4 antagonist KRH-1636 (1). A limited SAR study based on the three functionalities of 1 was first conducted, followed by site-directed mutagenesis studies. The receptor mapping showed that both the potency and affinity of 1 were dependent on the transmembrane residues His(113), Asp(171), Asp(262), and His(281) and also suggested the involvement of Tyr(45) and Gln(200) (potency) and Tyr(116) and Glu(288) (affinity). Molecular docking of 1 to an X-ray structure of CXCR4 showed that the l-Arg guanidino group of 1 forms polar interactions with His(113) and Asp(171) and the (pyridin-2-ylmethyl)amino moiety is anchored by Asp(262) and His(281), whereas the naphthalene ring is tightly packed in a hydrophobic subpocket formed by the aromatic side chains of Trp(94), Tyr(45), and Tyr(116). The detailed picture of ligand-receptor interactions provided here will assist in structure-based design and further development of small-molecule peptidomimetic CXCR4 antagonists.

Herein, we investigated whether G protein-coupled signaling via the vasopressin receptors of the V1a and V2 subtypes (V1aR and V2R) could be obtained as a direct response to hyperosmolar challenges and/or whether hyperosmolar challenges could augment classical vasopressin-dependent V1aR signaling. The V1aR-dependent response was monitored indirectly via its effects on aquaporin 4 (AQP4) when heterologously expressed in Xenopus oocytes and V1aR and V2R function was directly monitored following heterologous expression in COS-7 cells. A tendency toward an osmotically induced, V1aR-mediated reduction in AQP4-dependent water permeability was observed, although osmotic challenges failed to mimic vasopressin-dependent V1aR-mediated internalization of AQP4. Direct monitoring of inositol phosphate (IP) production of V1aR-expressing COS-7 cells demonstrated an efficient vasopressin-dependent response that was, however, independent of hyperosmotic challenges. Similarly, the cAMP production by the V2R was unaffected by hyperosmotic challenges although, in contrast to the V1aR, the V2R displayed an ability to support alternative signaling (IP production) at higher concentration of vasopressin. V1aR and V2R respond directly to vasopressin exposure, but they do not have an ability to act as osmo- or volume sensors when exposed to an osmotic gradient in the absence or presence of vasopressin., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The use of receptor-ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX3CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX3CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX3CL1, CX3CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo.

Peptide YY (PYY) is a 36 amino acid peptide hormone released from enteroendocrine cells. An N-terminally degraded metabolite, PYY3-36, has anorexigenic effects, which makes the PYY system a target for obesity treatment. However, little is known about the kinetics and degradation products of PYY. A related peptide, Neuropeptide Y (NPY), may be degraded from the C-terminus. We therefore investigated PYY degradation after in vitro incubations in porcine plasma and blood and in vivo by infusing PYY3-36 into multicatheterized pigs (n = 7) (2 pmol/kg/min). Plasma samples were analyzed by region-specific radioimmunoassays (RIA) and HPLC analysis. A metabolite, corresponding to PYY3-34 was formed after incubation in plasma and blood and during the infusion study. When taking the C-terminal degradation into account, the half-life (T½) of PYY in blood and plasma amounted to 3.4 ± 0.2 and 6.2 ± 0.2 h, respectively. After PYY3-36 infusion in pigs, the peptide was degraded with a T½ of 3.6 ± 0.5 min. Significant extraction (20.5 ± 8.0%) compatible with glomerular filtration was observed across the kidneys and significant C-terminal degradation (26.5 ± 4.8%) was observed across the liver. Net balances across the hind limb, splanchnic bed, and lungs were not significantly different from zero. PYY3-34 was unable to activate the Y2 receptor in a transfected cell line. In conclusion, PYY3-36 is extensively degraded to PYY3-34 in the pig, a degradation that renders the peptide inactive on the Y2 receptor. Currently used assays are unlikely to be able to detect this degradation and therefore measure falsely elevated levels of PYY3-36, leading to underestimation of its physiological effects., (© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Unlabelled: Coevolution of herpesviruses with their respective host has resulted in a delicate balance between virus-encoded immune evasion mechanisms and host antiviral immunity. BILF1 encoded by human Epstein-Barr virus (EBV) is a 7-transmembrane (7TM) G-protein-coupled receptor (GPCR) with multiple immunomodulatory functions, including attenuation of PKR phosphorylation, activation of G-protein signaling, and downregulation of major histocompatibility complex (MHC) class I surface expression. In this study, we explored the evolutionary and functional relationships between BILF1 receptor family members from EBV and 12 previously uncharacterized nonhuman primate (NHP) lymphocryptoviruses (LCVs). Phylogenetic analysis defined 3 BILF1 clades, corresponding to LCVs of New World monkeys (clade A) or Old World monkeys and great apes (clades B and C). Common functional properties were suggested by a high degree of sequence conservation in functionally important regions of the BILF1 molecules. A subset of BILF1 receptors from EBV and LCVs from NHPs (chimpanzee, orangutan, marmoset, and siamang) were selected for multifunctional analysis. All receptors exhibited constitutive signaling activity via G protein Gαi and induced activation of the NF-κB transcription factor. In contrast, only 3 of 5 were able to activate NFAT (nuclear factor of activated T cells); chimpanzee and orangutan BILF1 molecules were unable to activate NFAT. Similarly, although all receptors were internalized, BILF1 from the chimpanzee and orangutan displayed an altered cellular localization pattern with predominant cell surface expression. This study shows how biochemical characterization of functionally important orthologous viral proteins can be used to complement phylogenetic analysis to provide further insight into diverse microbial evolutionary relationships and immune evasion function., Importance: Epstein-Barr virus (EBV), known as an oncovirus, is the only human herpesvirus in the genus Lymphocryptovirus (LCV). EBV uses multiple strategies to hijack infected host cells, establish persistent infection in B cells, and evade antiviral immune responses. As part of EBV's immune evasion strategy, the virus encodes a multifunctional 7-transmembrane (7TM) G-protein-coupled receptor (GPCR), EBV BILF1. In addition to multiple immune evasion-associated functions, EBV BILF1 has transforming properties, which are linked to its high constitutive activity. We identified BILF1 receptor orthologues in 12 previously uncharacterized LCVs from nonhuman primates (NHPs) of Old and New World origin. As 7TM receptors are excellent drug targets, our unique insight into the molecular mechanism of action of the BILF1 family and into the evolution of primate LCVs may enable validation of EBV BILF1 as a drug target for EBV-mediated diseases, as well as facilitating the design of drugs targeting EBV BILF1., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Glucose-dependent insulinotropic polypeptide (GIP) is glucagonotropic, and glucagon-like peptide-1 (GLP-1) is glucagonostatic. We studied the effects of GIP and GLP-1 on glucagon responses to insulin-induced hypoglycemia in patients with type 1 diabetes mellitus (T1DM). Ten male subjects with T1DM (C-peptide negative, age [mean ± SEM] 26 ± 1 years, BMI 24 ± 0.5 kg/m(2), HbA1c 7.3 ± 0.2%) were studied in a randomized, double-blinded, crossover study, with 2-h intravenous administration of saline, GIP, or GLP-1. The first hour, plasma glucose was lowered by insulin infusion, and the second hour constituted a "recovery phase." During the recovery phase, GIP infusions elicited larger glucagon responses (164 ± 50 [GIP] vs. 23 ± 25 [GLP-1] vs. 17 ± 46 [saline] min ⋅ pmol/L, P < 0.03) and endogenous glucose production was higher with GIP and lower with GLP-1 compared with saline (P < 0.02). On the GIP days, significantly less exogenous glucose was needed to keep plasma glucose above 2 mmol/L (155 ± 36 [GIP] vs. 232 ± 40 [GLP-1] vs. 212 ± 56 [saline] mg ⋅ kg(-1), P < 0.05). Levels of insulin, cortisol, growth hormone, and noradrenaline, as well as hypoglycemic symptoms and cognitive function, were similar on all days. Our results suggest that during hypoglycemia in patients with T1DM, exogenous GIP increases glucagon responses during the recovery phase after hypoglycemia and reduces the need for glucose administration., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Being present in around 90% of the worldwide population, Epstein-Barr virus (EBV) is an exceptionally prevalent virus. This highly successful virus establishes a latent infection in resting memory B cells and is maintained in a balance between viral homeostasis on one side and antiviral defense of the immune system on the other side. The life cycle of EBV is dependent on many viral proteins, but EBV also regulates a number of endogenous proteins. 7TM receptors and ligands of viral and host origin are examples of such proteins. 7TM receptors are highly druggable and they are among the most popular class of investigational drug targets. The 7TM receptor encoded by EBV-BILF1, is known to downregulate cell surface MHC class I expression as part of the immune evasion strategy of EBV. However, the functional impact of the relationship between EBV and the regulated endogenous 7TM receptors and ligands is still unclear. This is for instance the case for the most upregulated 7TM receptor EBI2 (EBV-induced gene 2 or GPR183). Whereas some regulated genes have been suggested to be involved in the EBV life cycle, others could also be important for the antiviral immune defense. As many of these 7TM receptors and ligands have been shown to be modulated in EBV-associated diseases, targeting these could provide an efficient and specific way to inhibit EBV-associated disease progression. Here, we will review current knowledge on EBV infection, the immune defense against EBV and 7TM receptors and ligands being either encoded or manipulated by EBV., (© 2015 Elsevier Inc. All rights reserved.)

Over the last 5 years, X-ray structures of CXCR4 in complex with three different ligands (the small-molecule antagonist IT1t, the polypeptide antagonist CVX15 and the viral chemokine antagonist vMIP-II) have been released. In addition to the inherent scientific value of these specific X-ray structures, they provide a reliable structural foundation for studies of the molecular interactions between CXCR4 and its key peptide ligands (CXCL12 and HIV-1 gp120), and serve as valuable templates for further development of small-molecule CXCR4 antagonists with therapeutic potential. We here review recent computational studies of the molecular interactions between CXCR4 and its peptide ligands - based on the X-ray structures of CXCR4 - and the current status of small-molecule peptide and peptidomimetic CXCR4 antagonists.

Background and Purpose: The cyclopentapeptide FC131 (cyclo(-L-Arg(1) -L-Arg(2) -L-2-Nal(3) -Gly(4) -D-Tyr(5) -)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments., Experimental Approach: Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data., Key Results: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic pocket) respectively. Arg(1) forms charge-charge interactions with Asp(187) in ECL-2, while D-Tyr(5) points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu(288) via two water molecules. Intriguingly, Tyr(116) and Glu(288) form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity., Conclusions and Implications: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists., (© 2014 The British Pharmacological Society.)

The chemokine receptor CCR2 is a G protein-coupled receptor that is involved in many diseases characterized by chronic inflammation, and therefore a large variety of CCR2 small molecule antagonists has been developed. On the basis of their chemical structures these antagonists can roughly be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor approach to obtain insight into the binding site of the allosteric antagonists and additionally introduced eight single point mutations in CCR2 to further characterize the putative binding pocket. All constructs were studied in radioligand binding and/or functional IP turnover assays, providing evidence for an intracellular binding site for CCR2-RA-[R], JNJ-27141491, and SD-24. For CCR2-RA-[R] the most important residues for binding were found to be the highly conserved tyrosine Y(7.53) and phenylalanine F(8.50) of the NPxxYx(5,6)F motif, as well as V(6.36) at the bottom of TM-VI and K(8.49) in helix-VIII. These findings demonstrate for the first time the presence of an allosteric intracellular binding site for CCR2 antagonists. This contributes to an increased understanding of the interactions of diverse ligands at CCR2 and may allow for a more rational design of future allosteric antagonists., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Structure-activity relationship studies of the cyclopentapeptide CXCR4 antagonists (cyclo(-l-/d-Arg(1)-Arg(2)-2-Nal(3)-Gly(4)-d-Tyr(5)-)) suggest that the l-/d-Arg(1)-Arg(2)-2-Nal(3) tripeptide sequence contained within these cyclopentapeptides serves as a recognition motif for peptidic CXCR4 antagonists. Starting by dissecting the cyclopentapeptide structure and reintroducing cyclic constraints in a stepwise manner, we here report a novel class of scaffold-based tripeptidomimetic CXCR4 antagonists based on the d-Arg-Arg-2-Nal motif. Biological testing of the prototype compounds showed that they represent new peptidomimetic hits; importantly, the modular nature of the scaffold provides an interesting starting point for future ligand optimization., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The seven transmembrane G protein-coupled receptor Epstein-Barr virus (EBV) induced gene 2 (EBI2; also known as GPR183) was identified in 1993 on the basis of its substantial upregulation in EBV-infected cells. It is primarily expressed in lymphoid cells; most abundantly in B cells. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous EBI2 agonist is 7α, 25-dihydroxyxcholesterol (7α,25-OHC), yet many structurally related oxysterols can bind to an EBI2 pocket that is defined by the upper parts of the transmembrane helices and extracellular receptor regions. EBI2 signals via Gαi, as well as via G protein-independent pathways like β-arrestin recruitment. The concerted action of these pathways leads to cell migration. By genetically interfering with its up- and downregulation, EBI2 was also recently shown to induce cell proliferation, an action that could be inhibited by small molecule antagonists. Here, we focus on the oxysterol-EBI2 axis in immune control, including its role in the EBV life cycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonists and discuss EBI2 as therapeutic target for diseases of the immune system., (© 2014 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may not be absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro-switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of "classic" redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a single chemokine may bind to several receptors - in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles and different functional outcomes in a ligand-, receptor-, or cell/tissue-defined manner. As the low number of successful drug development plans implies, there are great difficulties in targeting chemokine receptors; in particular with regard to receptor antagonists as anti-inflammatory drugs. Un-defined and putative non-selective targeting of the complete cellular signaling system could be the underlying cause of lack of success. Therefore, biased ligands could be the solution.