Structural basis of ligand binding modes at the neuropeptide Y Y1 receptor.

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor superfamily and have important roles in food intake, anxiety and cancer biology 1,2 . The NPY-Y receptor system has emerged as one of the most complex networks with three peptide ligands (NPY, peptide YY and pancreatic polypeptide) binding to four receptors in most mammals, namely the Y1, Y2, Y4 and Y5 receptors, with different affinity and selectivity 3 . NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y1 receptor (Y1R) 4 . A number of peptides and small-molecule compounds have been characterized as Y1R antagonists and have shown clinical potential in the treatment of obesity 4 , tumour 1 and bone loss 5 . However, their clinical usage has been hampered by low potency and selectivity, poor brain penetration ability or lack of oral bioavailability 6 . Here we report crystal structures of the human Y1R bound to the two selective antagonists UR-MK299 and BMS-193885 at 2.7 and 3.0 Å resolution, respectively. The structures combined with mutagenesis studies reveal the binding modes of Y1R to several structurally diverse antagonists and the determinants of ligand selectivity. The Y1R structure and molecular docking of the endogenous agonist NPY, together with nuclear magnetic resonance, photo-crosslinking and functional studies, provide insights into the binding behaviour of the agonist and for the first time, to our knowledge, determine the interaction of its N terminus with the receptor. These insights into Y1R can enable structure-based drug discovery that targets NPY receptors.

Basal Histamine H4 Receptor Activation: Agonist Mimicry by the Diphenylalanine Motif.

Histamine H4 receptor (H4 R) orthologues are G-protein-coupled receptors (GPCRs) that exhibit species-dependent basal activity. In contrast to the basally inactive mouse H4 R (mH4 R), human H4 R (hH4 R) shows a high degree of basal activity. We have performed long-timescale molecular dynamics simulations and rigidity analyses on wild-type hH4 R, the experimentally characterized hH4 R variants S179M, F169V, F169V+S179M, F168A, and on mH4 R to investigate the molecular nature of the differential basal activity. H4 R variant-dependent differences between essential motifs of GPCR activation and structural stabilities correlate with experimentally determined basal activities and provide a molecular explanation for the differences in basal activation. Strikingly, during the MD simulations, F16945.55 dips into the orthosteric binding pocket only in the case of hH4 R, thus adopting the role of an agonist and contributing to the stabilization of the active state. The results shed new light on the molecular mechanism of basal H4 R activation that are of importance for other GPCRs.

Split luciferase-based assay for simultaneous analyses of the ligand concentration- and time-dependent recruitment of ß-arrestin2.

Functional selectivity of agonists has gained increasing interest in G protein-coupled receptor (GPCR) research, e.g. due to expectations of drugs with reduced adverse effects. Different agonist-dependent GPCR conformations are conceived to selectively activate a balanced or imbalanced intracellular signalling response, involving e.g. different Gα subtypes, Gßγ-subunits and ß-arrestins. To discriminate between the different signalling pathways (bias), sensitive techniques are needed that do not interfere with signalling. We applied split luciferase complementation to the GPCR/ß-arrestin2 interaction and thoroughly analysed the influence of its implementation on intracellular signalling. This led to an assay enabling the functional characterization of ligands at the hH1R, the hM1,5R and the hNTS1R in live HEK293T cells. As demonstrated at the hM1,5R, the assay was sensitive enough to identify iperoxo as a superagonist. Time-dependent analyses of the recruitment of ß-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with ß-arrestin2 and their recycling to the cell membrane. The developed ß-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and ß-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.

Alkyl derivatives of 1,3,5-triazine as histamine H4 receptor ligands.

This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H4 receptor (hH4R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH4R affinity with a Ki of 160 nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC50 = 32 nM) and [35S]GTPγS binding assay (pKb = 6.67). In addition, antinociceptive activity of 14in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).

Molecular Mechanisms of Biased and Probe-Dependent Signaling at CXC-Motif Chemokine Receptor CXCR3 Induced by Negative Allosteric Modulators.

Our recent explorations of allosteric modulators with improved properties resulted in the identification of two biased negative allosteric modulators, BD103 (N-1-{[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimi-din2yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl}]butanamide) and BD064 (5-[(N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl-2-[4-fluoro-3-(trifluoromethyl)phenyl]acetamido)methyl]-2-fluorophenyl}boronic acid), that exhibited probe-dependent inhibition of CXC-motif chemokine receptor CXCR3 signaling. With the intention to elucidate the structural mechanisms underlying their selectivity and probe dependence, we used site-directed mutagenesis combined with homology modeling and docking to identify amino acids of CXCR3 that contribute to modulator binding, signaling, and transmission of cooperativity. With the use of allosteric radioligand RAMX3 ([3H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide), we identified that F1313.32 and Y3087.43 contribute specifically to the binding pocket of BD064, whereas D1864.60 solely participates in the stabilization of binding conformation of BD103. The influence of mutations on the ability of negative allosteric modulators to inhibit chemokine-mediated activation (CXCL11 and CXCL10) was assessed with the bioluminescence resonance energy transfer-based cAMP and ß-arrestin recruitment assay. Obtained data revealed complex molecular mechanisms governing biased and probe-dependent signaling at CXCR3. In particular, F1313.32, S3047.39, and Y3087.43 emerged as key residues for the compounds to modulate the chemokine response. Notably, D1864.60, W2686.48, and S3047.39 turned out to play a role in signal pathway selectivity of CXCL10, as mutations of these residues led to a G protein-active but ß-arrestin-inactive conformation. These diverse effects of mutations suggest the existence of ligand- and pathway-specific receptor conformations and give new insights in the sophisticated signaling machinery between allosteric ligands, chemokines, and their receptors, which can provide a powerful platform for the development of new allosteric drugs with improved pharmacological properties.

Fluorescence- and Radiolabeling of [Lys4,Nle17,30]hPP Yields Molecular Tools for the NPY Y4 Receptor.

The neuropeptide Y (NPY) Y4 receptor (Y4R) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the Y4R were reported. Previously, [Lys4]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ε-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys4]hPP by norleucine and prepared the corresponding [3H]propionylated ([3H]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y1, Y2, Y4, and Y5 receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) Y4R assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition Y4R binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [3H]12 affinity (Kd in Na+-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and Y4R-mediated internalization of 13 (Kd in Na+-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [3H]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na+-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na+ on Y5R affinity became obvious, when the radioligands [H]12 and [3H]propionyl-pNPY were investigated in saturation binding and competition binding.

Topotecan-induced ABCG2 expression in MCF-7 cells is associated with decreased CD24 and EpCAM expression and a loss of tumorigenicity.

Human breast cancer shows a considerable heterogeneity regarding the expression of CD24, CD44, EpCAM, and HER2. These markers are involved in cell adhesion, migration, and proliferation, and thus affect metastasis and, in turn, patient's outcome. The ATP-driven efflux pump (ABC transporter) breast cancer resistance protein (BCRP, ABCG2) is known to confer resistance to a wide variety of structurally unrelated cytostatics and defines subpopulations with enhanced tumor-initiating capacity. The expression of ABCG2 can be induced by treatment with different cytostatic drugs. Concurrent effects of such treatments on the expression of the aforementioned marker proteins and cellular properties related to cancer-initiating cells have not been examined thoroughly. Here, we investigated the effect of the ABCG2 substrate topotecan on the MCF-7 breast cancer cell line and analyzed CD24, CD44, EpCAM, and HER2 expression by flow cytometry. Moreover, we examined the impact of topotecan treatment on the sphere-forming ability in vitro and the tumorigenicity in immunodeficient NMRI-nu/nu and NSG mice. We found an elevated ABCG2 expression in MCF-7 cells in the presence of 500 nM topotecan. Compared with untreated MCF-7 cells, the application of topotecan induced a subpopulation with decreased CD24/EpCAM expression, whereas CD44 expression remained largely unchanged. Topotecan-treated cells showed an impaired mammosphere formation capacity in vitro and reduced tumorigenicity in immunodeficient mice. The data indicate that ABCG2 induction is not necessarily linked to increased tumorigenicity and suggest a major role of CD24 and EpCAM for the preservation of self-renewal capacity in MCF-7 cells and tumor outgrowth in vivo.

M2 Subtype preferring dibenzodiazepinone-type muscarinic receptor ligands: Effect of chemical homo-dimerization on orthosteric (and allosteric?) binding.

A series of new dibenzodiazepinone-type muscarinic receptor ligands, including two homo-dimeric compounds, was prepared. Sixteen representative compounds were characterized in equilibrium binding studies with [(3)H]N-methylscopolamine ([(3)H]NMS) at the muscarinic receptor subtype M2, and seven selected compounds were additionally investigated at M1, M3, M4 and M5 with respect to receptor subtype selectivity. The side chain of the known M2 preferring muscarinic receptor antagonist DIBA was widely varied with respect to chain length and type of the basic group (amine, imidazole, guanidine and piperazine). Most of the structural changes were well tolerated with respect to muscarinic receptor binding, determined by displacement of [(3)H]NMS. Compounds investigated at all subtypes shared a similar selectivity profile, which can be summarized as M2>M1≈M4>M3≈M5 (46, 50, 57, 62-64) and M2>M1≈M4>M3>M5 (1, 58). The homo-dimeric dibenzodiazepinone derivatives UNSW-MK250 (63) and UNSW-MK262 (64) exhibited the highest M2 receptor affinities (pIC50=9.0 and 9.2, respectively). At the M2 receptor a steep curve slope of -2 was found for the dimeric ligand 63, which cannot be described according to the law of mass action, suggesting a more complex mechanism of binding. In addition to equilibrium binding studies, for selected ligands, we determined pEC50,diss, an estimate of affinity to the allosteric site of M2 receptors occupied with [(3)H]NMS. Compounds 58 and 62-64 were capable of retarding [(3)H]NMS dissociation by a factor >10 (Emax,diss >92%), with highest potency (pEC50,diss=5.56) residing in the dimeric compound 64. As the monomeric counterpart of 64 was 100 times less potent (62: pEC50,diss=3.59), these data suggest that chemical dimerization of dibenzodiazepinone-type M receptor ligands can enhance allosteric binding.

Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists.

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [(35)S]GTPγS assays on recombinant human and guinea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in radioligand competition binding studies. H2R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes.

Toward Labeled Argininamide-Type NPY Y1 Receptor Antagonists: Identification of a Favorable Propionylation Site in BIBO3304.

Aiming at molecular tools for the neuropeptide Y Y1 receptor (Y1 R), three types of derivatives of the argininamide-type Y1 R antagonist BIBO3304 were prepared by (i) propionylation at the guanidine group (3), (ii) substitution at the urea moiety with a propionamidobutyl residue (4), and (iii) replacement of ureidomethyl by a propionylaminomethyl group (5). With Ki and Kb values in the range of 1.5-4.3 nM, determined in binding and functional assays, and high selectivity for the Y1 R over the Y2 R, Y4 R, and Y5 R, compounds 4 and 5 were identified as promising candidates for radiolabeling by [(3) H]propionylation according to established protocols.

Autodisplay of Human Hyaluronidase Hyal-1 on Escherichia coli and Identification of Plant-Derived Enzyme Inhibitors.

Hyaluronan (HA) is the main component of the extracellular matrix (ECM). Depending on its chain size, it is generally accepted to exert diverse effects. High molecular weight HA is anti-angiogenic, immunosuppressive and anti-inflammatory, while lower fragments are angiogenic and inflammatory. Human hyaluronidase Hyal-1 (Hyal-1) is one of the main enzymes in the metabolism of HA. This makes Hyal-1 an interesting target. Not only for functional and mechanistic studies, but also for drug development. In this work, Hyal-1 was expressed on the surface of E. coli, by applying Autodisplay, to overcome formation of inactive "inclusion bodies". With the cells displaying Hyal-1 an activity assay was performed using "stains-all" dye. Subsequently, the inhibitory effects of four saponins and 14 plant extracts on the activity of surface displayed Hyal-1 were evaluated. The determined IC50 values were 177 µM for glycyrrhizic acid, 108 µM for gypsophila saponin 2, 371 µM for SA1657 and 296 µM for SA1641. Malvae sylvestris flos, Equiseti herba and Ononidis radix extracts showed IC50 values between 1.4 and 1.7 mg/mL. In summary, Autodisplay enabled the expression of functional human target protein Hyal-1 in E. coli and facilitated an accelerated testing of potential inhibitors.

No evidence for histamine H4 receptor in human monocytes.

The histamine H4 receptor (H4R) is a classic pertussis toxin-sensitive Gi protein-coupled receptor that mediates increases in intracellular calcium concentration ([Ca(2+)]i). The presence of H4R in human eosinophils has been rigorously documented by several independent groups. It has also been suggested that H4R is expressed in human monocytes, but this suggestion hinges in part on H4R antibodies with questionable specificity. This situation prompted us to reinvestigate H4R expression in human monocytes. As positive control, we studied human embryonic kidney 293T cells stably expressing the human H4R (hH4R). In these cells, histamine (HA) and the H4R agonist UR-PI376 (2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine) induced pertussis toxin-sensitive [Ca(2+)]i increases. However, in quantitative real-time polymerase chain reaction studies we failed to detect hH4R mRNA in human monocytes and U937 promonocytes. In human monocytes, ATP and N-formyl-l-methionyl-l-leucyl-l-phenylalanine increased [Ca(2+)]i, but HA, UR-PI376, and 5-methylhistamine (a dual H4R/H2 receptor agonist) did not. In U937 promonocytes and differentiated U937 cells, HA increased [Ca(2+)]i, but this increase was mediated via HA H1 receptor. In conclusion, there is no evidence for the presence of H4R in human monocytes.

Synthesis and functional characterization of imbutamine analogs as histamine H3 and H4 receptor ligands.

Imbutamine (4-(1H-imidazol-4-yl)butanamine) is a potent histamine H3 (H3R) and H4 receptor (H4R) agonist (EC50 values: 3 and 66 nM, respectively). Aiming at improved selectivity for the H4R, the imidazole ring in imbutamine was methyl-substituted or replaced by various differently substituted heterocycles (1,2,3-triazoles, 1,2,4-triazoles, pyridines, pyrimidines) as potential bioisosteres. Investigations in [(35)S]GTPγS binding assays using membranes of Sf9 insect cells expressing the respective human histamine receptor subtype revealed only very weak activity of most of the synthesized hetarylalkylamines at both receptors. By contrast, the introduction of substituents at the 4-imidazolyl ring was most effective regarding H4R selectivity. This holds for methyl substitution in position 2 and, especially, in position 5. 5-Methylimbutamine (H4R: EC50 = 59 nM, α = 0.8) was equipotent with imbutamine at the hH4R, but revealed about 16-fold selectivity for the hH4R compared to the hH3R (EC50 980 nM, α = 0.36), whereas imbutamine preferred the hH3R. The functional activities were in agreement with radioligand binding data. The results support the hypothesis that, by analogy with histamine, methyl substitution in histamine homologs offers a way to shift the selectivity in favor of the H4R.

Synthesis and pharmacological characterization of new tetrahydrofuran based compounds as conformationally constrained histamine receptor ligands.

A series of tetrahydrofuran based compounds with a bicyclic core that provides conformational restriction were synthesized and investigated by radioligand displacement studies and functional [(35)S]GTPγS binding assays at the human histamine receptor (hHR) subtypes. The amines and ((1S,3R,5S,6R)- and ((1S,3S,5S,6R)-3-(1H-imidazol-5-yl)-2-oxabicyclo[3.1.0]hexan-6-yl)methanamine), exhibited submicromolar Ki values at the hH3R with 10-fold higher affinities than their corresponding (6S)-epimers and 25- and >34-fold selectivity over the hH4R, respectively. Both compounds act as neutral antagonists at the hH3R with KB values of 181 and 32 nM, respectively. The cyanoguanidines of the imidazole series and the oxazole analogues turned out to be inactive at all hHR subtypes.

Dimeric argininamide-type neuropeptide Y receptor antagonists: chiral discrimination between Y1 and Y4 receptors.

The structurally related peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP) are endogenous agonists of the NPY receptor (YR) family, which in humans comprises four functionally expressed subtypes, designated Y1R, Y2R, Y4R and Y5R. Nonpeptide antagonists with high affinity and selectivity have been described for the Y1R, Y2R and Y5R, but such compounds are still lacking for the Y4R. In this work, the structures of the high affinity selective (R)-argininamide-type Y1R antagonists BIBP3226 and BIBO3304 were linked via the guanidine or urea moieties to give homo-dimeric argininamides with linker lengths ranging from 31 to 41 atoms. Interestingly, the twin compounds proved to be by far less selective for the Y1R than the R-configured monovalent parent compounds. The decrease in selectivity ratio was most pronounced for Y1R versus Y4R subtype, resulting in comparable affinities of bivalent ligands for Y1R and Y4R (e.g. UR-MK177 ((R,R)-49): Ki=230nM (Y1R) and 290nM (Y4R)). With a Ki value of 130nM and a Kb value of 20nM, UR-MK188 ((R,R)-51) was superior to all Y4R antagonists known to date. The S,S-configured optical antipodes of UR-MK177 and UR-MK188 (UR-MEK381 ((S,S)-49) and UR-MEK388 ((S,S)-51)) were synthesized to investigate the stereochemical discrimination by the different receptor subtypes. Whereas preference for R,R-configured argininamides was characteristic of the Y1R, stereochemical discrimination by the Y4R was not observed. This may pave the way to selective Y4R antagonists.

G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake.

More selective interactions of nanoparticles with cells would substantially increase their potential for diagnostic and therapeutic applications. Thus, it would not only be highly desirable that nanoparticles can be addressed to any cell with high target specificity and affinity, but that we could unequivocally define whether they rest immobilized on the cell surface as a diagnostic tag, or if they are internalized to serve as a delivery vehicle for drugs. To date no class of targets is known that would allow direction of nanoparticle interactions with cells alternatively into one of these mutually exclusive events. Using MCF-7 breast cancer cells expressing the human Y(1)-receptor, we demonstrate that G protein-coupled receptors provide us with this option. We show that quantum dots carrying a surface-immobilized antagonist remain with nanomolar affinity on the cell surface, and particles carrying an agonist are internalized upon receptor binding. The receptor functions like a logic "and-gate" that grants cell access only to those particles that carry a receptor ligand "and" where the ligand is an agonist. We found that agonist- and antagonist-modified nanoparticles bind to several receptor molecules at a time. This multiligand binding leads to five orders of magnitude increased-receptor affinities, compared with free ligand, in displacement studies. More than 800 G protein-coupled receptors in humans provide us with the paramount advantage that targeting of a plethora of cells is possible, and that switching from cell recognition to cell uptake is simply a matter of nanoparticle surface modification with the appropriate choice of ligand type.

Synthesis and dual histamine H1 and H2 receptor antagonist activity of cyanoguanidine derivatives.

Premedication with a combination of histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists has been suggested as a prophylactic principle, for instance, in anaesthesia and surgery. Aiming at pharmacological hybrids combining H1R and H2R antagonistic activity, a series of cyanoguanidines 14-35 was synthesized by linking mepyramine-type H1R antagonist substructures with roxatidine-, tiotidine-, or ranitidine-type H2R antagonist moieties. N-desmethylmepyramine was connected via a poly-methylene spacer to a cyanoguanidine group as the "urea equivalent" of the H2R antagonist moiety. The title compounds were screened for histamine antagonistic activity at the isolated ileum (H1R) and the isolated spontaneously beating right atrium (H2R) of the guinea pig. The results indicate that, depending on the nature of the H2R antagonist partial structure, the highest H1R antagonist potency resided in roxatidine-type compounds with spacers of six methylene groups in length (compound 21), and tiotidine-type compounds irrespective of the alkyl chain length (compounds 28, 32, 33), N-cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[2-[N-[2-[N-(4-methoxybenzyl)-N-(pyridyl)-amino] ethyl]-N-methylamino]ethyl] guanidine (25, pKB values: 8.05 (H1R, ileum) and 7.73 (H2R, atrium) and the homologue with the mepyramine moiety connected by a six-membered chain to the tiotidine-like partial structure (compound 32, pKB values: 8.61 (H1R) and 6.61 (H2R) were among the most potent hybrid compounds. With respect to the development of a potential pharmacotherapeutic agent, structural optimization seems possible through selection of other H1R and H2R pharmacophoric moieties with mutually affinity-enhancing properties.

Novel azulene derivatives for the treatment of erectile dysfunction.

Based on the dopamine D(4) receptor partial agonist FAUC 3019, a series of azulenylmethylpiperazines was synthesized and affinities for the monoaminergic GPCRs including dopamine, serotonin, histamine and α-adrenergic receptor subtypes were determined. Ligand efficacies of the most promising test compounds revealed the N,N-dimethylaminomethyl substituted azulene 11 to be the most potent D(4) partial agonist (EC(50)=0.41 nM). This candidate was investigated for its ability to promote penile erection. Applying an in vivo animal model, test compound 11 turned out to stimulate penile erection in male rats with superior potency in low concentrations when compared to apomorphine.

Fast counter-electroosmotic capillary electrophoresis-time-of-flight mass spectrometry of hyaluronan oligosaccharides.

Fast capillary electrophoresis-mass spectrometry measurements under counter-electroosmotic analyte migration conditions are presented. Efficient separations of a homologous series of six hyaluronan oligosaccharides (comprising 1-6 hyalobiuronic acid moieties) could be completed in 65 s. Separations were achieved in short-length fused silica capillaries under high electric field strengths of up to 1.25 kV·cm(-1). Capillary inner diameters ranging from 5 to 50 µm were investigated, resulting in an optimal value of 15 µm. The influence of capillary dimensions and buffer composition on separation efficiency and sensitivity are discussed. Optimal separations were achieved using a 28 cm × 15 µm capillary, a separation high voltage of 35 kV, a background electrolyte of 25 mM ammonium acetate adjusted to pH 8.5, and negative ionization mode. The optimized method was successfully applied to a bovine testicular hyaluronidase digest of hyaluronan. Only minimal sample pretreatment for protein-containing samples is required. The simple manual injection procedure and fast separations allow for a sample throughput of 35 samples per hour.

Paradoxical stimulatory effects of the "standard" histamine H4-receptor antagonist JNJ7777120: the H4 receptor joins the club of 7 transmembrane domain receptors exhibiting functional selectivity.

The histamine H(4) receptor (H(4)R) is expressed in several cell types of the immune system and is assumed to play an important pro-inflammatory role in various diseases, including bronchial asthma, atopic dermatitis, and pruritus. Accordingly, H(4)R antagonists have been suggested to provide valuable drugs for the treatment of these diseases. Over the past decade, the indole derivative 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has become the "standard" H(4)R antagonist and has been extensively used to assess the pathophysiological role of the H(4)R. However, the situation has now become more complicated by recent data (p. 749 and Naunyn Schmiedebergs Arch Pharmacol doi: 10.1007/s00210-011-0612-3) showing that JNJ7777120 can also activate ß-arrestin in a supposedly G(i)-protein-independent (pertussis toxin-insensitive) manner and that at certain H(4)R species orthologs, JNJ7777120 exhibits partial agonist efficacy with respect to G(i)-protein activation (steady-state high-affinity GTPase activity). These novel findings can be explained within the concept of functional selectivity or biased signaling, assuming unique ligand-specific receptor conformations with distinct signal transduction capabilities. Thus, great caution must be exerted when interpreting in vivo effects of JNJ7777120 as H(4)R antagonism. We discuss future directions to get out of the current dilemma in which there is no "standard" H(4)R antagonist available to the scientific community.