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3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H2 receptor (H2R) agonists, but their applicability as pharmacological tools to elucidate the largely unknown H2R functions in the central nervous system (CNS) is compromised by their concomitant high affinity toward dopamine D2-like receptors (especially to the D3R). To improve the selectivity, a series of novel carbamoylguanidine-type ligands containing various heterocycles, spacers, and side residues were rationally designed, synthesized, and tested in binding and/or functional assays at H1-4 and D2long/3 receptors. This study revealed a couple of selective candidates (among others 31 and 47), and the most promising ones were screened at several off-target receptors, showing good selectivities. Docking studies suggest that the amino acid residues (3.28, 3.32, E2.49, E2.51, 5.42, and 7.35) are responsible for the different affinities at the H2- and D2long/3-receptors. These results provide a solid base for the exploration of the H2R functions in the brain in further studies.

Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH(2)R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27) was synthesized and characterized. The highest hH(2)R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pK(i): 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pK(i): 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK(d) values comparable to the pK(i) values. The fluorescent probes 21, 23, and 25 could be used to localize H-2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Comprehensively characterized fluorescent probes for the histamine H3 receptor (H3R) and especially for the H4R orthologs [e.g., human (h) and mouse (m)] are highly needed as versatile complementar...

Histamine H4 receptor (H4R) orthologues are G‐protein‐coupled receptors (GPCRs) that exhibit species‐dependent basal activity. In contrast to the basally inactive mouse H4R (mH4R), human H4R (hH4R) shows a high degree of basal activity. We have performed long‐timescale molecular dynamics simulations and rigidity analyses on wild‐type hH4R, the experimentally characterized hH4R variants S179M, F169V, F169V+S179M, F168A, and on mH4R to investigate the molecular nature of the differential basal activity. H4R 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 hH4R, 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 H4R activation that are of importance for other GPCRs., How does it work? In addition to ligand‐dependent G‐protein‐coupled receptor (GPCR) activation, which has been studied intensively, GPCRs can also be activated spontaneously in the absence of a ligand. This study highlights the mechanisms behind such basal GPCR activation by using the example of the H4R, a GPCR with extraordinarily high basal activity.

Differences in sequence homology between human (h), mouse (m), and rat (r) histamine H4 receptors (H4R) cause discrepancies regarding affinities, potencies, and/or efficacies of ligands and therefore compromise translational animal models and the applicability of radioligands. Aiming at a radioligand enabling robust and comparative binding studies at the h/m/rH4Rs, 2,4-diaminopyrimidines were synthesized and pharmacologically investigated. The most notable compounds identified were two (partial) agonists with comparable potencies at the h/m/rH4Rs: UR-DEBa148 (N-neopentyl-4-(1,4,6,7-tetrahydro-5H-imidazo[4,5-c]pyridin-5-yl)pyrimidin-2-amine bis(2,2,2-trifluoroacetate), 43), the most potent [pEC50 (reporter gene assay) = 9.9/9.6/10.3] compound in the series being slightly G-protein biased and UR-DEBa176 [(R)-4-[3-(dimethylamino)pyrrolidin-1-yl]-N-neopentylpyrimidin-2-amine bis(2,2,2-trifluoroacetate), 46, pEC50 (reporter gene assay) = 8.7/9.0/9.2], a potential "cold" form of a tritiated H4R ligand. After radiolabeling, binding studies with [3H]UR-DEBa176 ([3H]46) at the h/m/rH4Rs revealed comparable Kd values (41/17/22 nM), low nonspecific binding (11-17%, ∼Kd), and fast associations/dissociations (25-30 min) and disclosed [3H]UR-DEBa176 as useful molecular tool to determine h/m/rH4R binding affinities for H4R ligands.

New classes of alkylated hetarylpropylguanidines with different functionality and variation in spacer length were synthesized to determine their behavior at the four histamine receptor (H1R, H2R, H3R, H4R) subtypes. Alkylated guanidines with different terminal functional groups and varied basicity, like amine, guanidine and urea were developed, based on the lead structure SK&F 91486 (2). Furthermore, heteroatomic exchange at the guanidine structure of 2 led to simple analogues of the lead compound. Radioassays at all histamine receptor subtypes were accomplished, as well as organ bath studies at the guinea pig (gp) ileum (gpH1R) and right atrium (gpH2R). Ligands with terminal functionalization led to, partially, highly affine and potent structures (two digit nanomolar), which showed up a bad selectivity profile within the histamine receptor family. While the benzoylurea derivative 144 demonstrated a preference towards the human (h) H3R, S‐methylisothiourea analogue 143 obtained high affinity at the hH4R (pKi=8.14) with moderate selectivity. The molecular basis of the latter finding was supported by computational studies.

3-(2-Amino-4-methylthiazol-5-yl)propyl-substituted carbamoylguanidines are potent, subtype-selective histamine H

Even today, the role of the histamine H

Comprehensively characterized fluorescent probes for the histamine H

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GP CR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

[Image: see text] ABC transporters, including ABCG2, play a vital role in defending the human body against the vast range of xenobiotics. Even though this is beneficial for human health, these protein transporters have been implicated in the emerging resistance of cancer cells to a variety of structurally and functionally diverse anticancer drugs. In order to investigate their role in resistance, potent and selective ABCG2 modulators have been described in the literature. A leading class of modulators are the tariquidar analogues; however, their susceptibility to hydrolysis limits their applicable use. To overcome this, we synthesized a novel series of chalcone- and ketone-based compounds inspired by reported tariquidar analogues. Compounds were characterized and evaluated for their ABCG2 modulatory activity and ABC transporter selectivity. When compared to transporters ABCB1 and ABCC1, the chalcone-based compounds exhibited selectivity for ABCG2, while the ketone-based compounds showed only a slight preference for ABCG2. From the former series, chalcone 16d (UR-DP48) displayed similar activity to the reference fumitremorgin C, both producing comparable maximal effects. The compound exhibited marked antiproliferative activity, while cytotoxicity was less pronounced for the most active compound 17f from the ketone series. Chalcone-containing tariquidar analogues are promising modulators to aid in functional investigations of ABCG2 transporters.

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 Y-1, Y-2, Y-4 and Y-5 receptors, with different affinity and selectivity(3). NPY is the most powerful stimulant of food intake and this effect is primarily mediated by the Y-1 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 angstrom 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.

The cross-linked pentapeptides (2R, 7R)-diaminooctanedioyl- bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2R, 7R)BVD- 74D, (2R, 7R)-1) and octanedioyl-bis(Tyr-Arg-Leu-ArgTyr- amide) (2) as well as the pentapeptide Ac-Tyr-Arg-LeuArg- Tyr-amide (3) were previously described as neuropeptide Y Y-4 receptor (Y4R) partial agonists. Here, we report on a series of analogues of (2R, 7R)-1 and 2 in which Arg(2), Leu(3), or Arg(4) were replaced by the respective aza-amino acids. The replacement of Arg(2) in 3 with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr-N-omega-[(4-aminobutyl) aminocarbonyl] Arg-Leu-Arg-Tyr-amide (35), which was used as a precursor for a D-amino acid scan. The target compounds were investigated for Y4R functional activity in assays with complementary readouts: aequorin Ca2+ and beta-arrestin 1 or beta-arrestin 2 assays. In contrast to the parent compounds, which are Y4R agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide Y4R antagonists.

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 e-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 equil...

The neuropeptide Y (NPY) Y1 receptor (Y1R) selective radioligand (R)-Nα-(2,2-diphenylacetyl)-Nω-[4-(2-[18F]fluoropropanoylamino)butyl]aminocarbonyl-N-(4-hydroxybenzyl)argininamide ([18F]23), derived from the high-affinity Y1R antagonist BIBP3226, was developed for imaging studies of Y1R-positive tumors. Starting from the argininamide core bearing amine-functionalized spacer moieties, a series of fluoropropanoylated and fluorobenzoylated derivatives was synthesized and studied for Y1R affinity. The fluoropropanoylated derivative 23 displayed high affinity (Ki = 1.3 nM) and selectivity toward Y1R. Radiosynthesis was accomplished via 18F-fluoropropanoylation, yielding [18F]23 with excellent stability in mice; however, the biodistribution study revealed pronounced hepatobiliary clearance with high accumulation in the gall bladder (>100 %ID/g). Despite the unfavorable biodistribution, [18F]23 was successfully used for imaging of Y1R positive MCF-7 tumors in nude mice. Therefore, we suggest [18F]23 as a lead fo...

[Image: see text] Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor–ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH(2)R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19–27) was synthesized and characterized. The highest hH(2)R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19–21 (pK(i): 7.71–7.76) and cyanine labeled antagonists 23 and 25 (pK(i): 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry, and high content imaging. Saturation binding experiments revealed pK(d) values comparable to the pK(i) values. The fluorescent probes 21, 23, and 25 could be used to localize H(2) receptors in HEK cells and to determine the binding affinities of unlabeled compounds.

Tariquidar derivatives have been described as potent and selective ABCG2 inhibitors. However, their susceptibility to hydrolysis limits their applicability. The current study comprises the synthesis and characterization of novel tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in our previous tariquidar analog UR-ME22-1 (9). CuAAC (“click” reaction) gave convenient access to a triazole core as a substitute for the labile amide group and the labile ester moiety was replaced by different acyl groups in a Sugasawa reaction. A stability assay proved the enhancement of the stability in blood plasma. Compounds UR-MB108 (57) and UR-MB136 (59) inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM and belong to the most potent ABCG2 inhibitors described so far. Compound 57 was highly selective, whereas its PEGylated analog 59 showed some potency at ABCB1. Both 57 and 59 produced an ABCG2 ATPase-depressing effect which is in agreement with our precedent cryo-EM study identifying 59 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation. Thermostabilization of ABCG2 by 57 and 59 can be taken as a hint to comparable binding to ABCG2. As reference substances, compounds 57 and 59 allow additional mechanistic studies on ABCG2 inhibition. Due to their stability in blood plasma, they are also applicable in vivo. The highly specific inhibitor 57 is suited for PET labeling, helping to further elucidate the (patho)physiological role of ABCG2, e.g. at the BBB., European Journal of Medicinal Chemistry, 191, ISSN:0223-5234, ISSN:1768-3254

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [(35)S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R,100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([(35)S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.

// Lena Edalat 1, 2, * , Benjamin Stegen 2, * , Lukas Klumpp 2, 5 , Erik Haehl 2 , Karin Schilbach 3 , Robert Lukowski 1 , Matthias Kuhnle 4 , Gunther Bernhardt 4 , Armin Buschauer 4 , Daniel Zips 2 , Peter Ruth 1 , Stephan M. Huber 2 1 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tubingen, Tubingen, Germany 2 Department of Radiation Oncology, University of Tubingen, Tubingen, Germany 3 Department of General Pediatrics, Oncology/Hematology, University of Tubingen, Tubingen, Germany 4 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, Regensburg, Germany 5 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tubingen, Tubingen, Germany * These authors contributed equally to this work Correspondence to: Peter Ruth, e-mail: peter.ruth@uni-tuebingen.de Stephan M. Huber, e-mail: stephan.huber@uni-tuebingen.de Keywords: glioma, radiation therapy, patch-clamp recording, fura-2 Ca2 + imaging, transfilter migration Received: October 20, 2015 Accepted: January 29, 2016 Published: February 16, 2016 ABSTRACT Infiltration of the brain by glioblastoma cells reportedly requires Ca 2+ signals and BK K + channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca 2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca 2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo . Importantly, paxilline blocked IR-induced migration in vivo . Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K + channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo . This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.

Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

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 14 in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).

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, Gβγ-subunits and β-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/β-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 β-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with β-arrestin2 and their recycling to the cell membrane. The developed β-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and β-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.

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 H

The earlier an activation of a G protein-dependent signalling cascade at a G protein-coupled receptor (GPCR) is probed, the less amplificatory effects contribute to the measured signal. This is especially useful in case of a precise quantification of agonist efficacies, and is of paramount importance, when determining agonist bias in relation to the β-arrestin pathway. As most canonical assays with medium to high throughput rely on the quantification of second messengers, and assays affording more proximal readouts are often limited in throughput, we developed a technique with a proximal readout and sufficiently high throughput that can be used in live cells. Split luciferase complementation (SLC) was applied to assess the interaction of Gαq with its effector phospholipase C-β3. The resulting probe yielded an excellent Z’ value of 0.7 and offers a broad and easy applicability to various Gαq-coupling GPCRs (hH1R, hM1,3,5R, hNTS1R), expressed in HEK293T cells, allowing the functional characterisation of agonists and antagonists. Furthermore, the developed sensor enabled imaging of live cells by luminescence microscopy, as demonstrated for the hM3R. The versatile SLC-based probe is broadly applicable e.g. to the screening and the pharmacological characterisation of GPCR ligands as well as to molecular imaging.

Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field.

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.

Aiming at molecular tools for the neuropeptide Y Y1 receptor (Y1R), three types of derivatives of the argininamide-type Y1R antagonist BIBO3304 were prepared by (1) propionylation at the guanidine group (3), (2) substitution at the urea moiety with a propionamidobutyl residue (4) and (3) 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 Y1R over the Y2R, Y4R and Y5R, compounds 4 and 5 were identified as promising candidates for radiolabeling by [3H]propionylation according to established protocols.

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.

High performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) was optimized for the analysis of oligosaccharides derived from the extracellular matrix component hyaluronan. Using this sensitive approach, the separation of oligosaccharides consisting of two (molecular weight ca. 0.8 kDa) up to 25-30 (molecular weight: ca. 9.5-11.4 kDa) disaccharide moieties was possible. Standard oligosaccharides (comprising 2-4 repetitive disaccharides) were detectable at very low amounts of 0.2-0.3 pmol (20-30 nM). Including 10 min of column equilibration, a complex mixture of low molecular weight hyaluronan can be analyzed within 40 min. The HPAEC method was successfully applied to the study of the size-dependency of both the action of bovine testicular hyaluronidase (BTH) and the precipitation of hyaluronan by cetyltrimethylammonium bromide (CTAB), a physicochemical reaction often used for the determination of hyaluronan and hyaluronidase activity.

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 β-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 β-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.

The neuropeptide Y (NPY) Y

B(0)AT2, encoded by the SLC6A15 gene, is a transporter for neutral amino acids that has recently been implicated in mood and metabolic disorders. It is predominantly expressed in the brain, but little is otherwise known about its function. To identify inhibitors for this transporter, we screened a library of 3133 different bioactive compounds. Loratadine, a clinically used histamine H1 receptor antagonist, was identified as a selective inhibitor of B(0)AT2 with an IC50 of 4 μM while being less active or inactive against several other members of the SLC6 family. Reversible inhibition of B(0)AT2 was confirmed by electrophysiology. A series of loratadine analogues were synthesized to gain insight into the structure-activity relationships. Our studies provide the first chemical tool for B(0)AT2.

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.

Background and purpose Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4 R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4 R) and rat H4 receptor (rH4 R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4 R-F169V, mH4 R-V171F, hH4 R-S179A, hH4 R-S179M; double mutants: hH4 R-F169V+S179A, hH4 R-F169V+S179M and mH4 R-V171F+M181S. Experimental approach Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gβ1 γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([(3) H]-histamine), and in functional [(35) S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. Key results Constitutive activity decreased from the hH4 receptor via the hH4 R-F169V mutant to the hH4 R-F169V+S179A and hH4 R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4 R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. Conclusions and implications F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. Linked articles This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc.

The biodistribution of nanoparticles is a major subject of current nanomedical research. To date, however, the exact investigation of nanoparticle fate in the microenvironment of a main excretory organ, the kidney has largely been neglected. In this study, the biodistribution of polyethylene glycol-coated quantum dots (Qdots) with special focus on their interaction with the kidney is investigated. Upon intravenous injection, nanoparticles showed effective blood circulation in mice and significant renal accumulation after two hours. Histological analysis of the kidney revealed that Qdots were strongly associated to the intraglomerular mesangial cells. This preferential deposition of nanoparticles in the kidney mesangium is highly promising, since it could be of utmost value for site-specific treatment of severe kidney diseases like diabetic nephropathy in the future.

In the search for potential bioisosteres of the 4-imidazolyl ring in acylguanidines (e.g. UR-AK24), known to possess affinity to several histamine receptor subtypes (HxR, x = 1–4), and cyanoguanidine-type H4R agonists (e.g. UR-PI376), the contribution of various heterocycles to agonism, antagonism and HR subtype selectivity was studied (recombinant human H1,2,3,4Rs, isolated guinea pig organs (H1R, H2R)). While minor structural modifications of UR-PI376 analogues were not tolerated regarding H4R agonism, in the case of the acylguanidines, a 1,2,4-triazole ring shifted the selectivity toward the H2R.

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.

ABC, it's easy as 1 2 3! Bioisosteric replacement of the anilide core by an indole moiety considerably increased stability and gave potent and selective ABCG2 (BCRP) inhibitors. Some compounds are superior to the reference substances fumitremorgin C and Ko143 in terms of potency and efficacy and are the most potent ABCG2 modulators reported so far.