World ocean review: Mit den Meeren leben 5. Die Küsten - ein wertvoller Lebensraum unter Druck

Die fünfte Ausgabe des „World Ocean Review“ (WOR) beschäftigt sich mit dem Lebensraum Küste und den vielfältigen Erwartungen, die an diesen Lebensraum gestellt werden. Der WOR 5 gibt einen Einblik in die über Jahrmillionen zurückreichende Geschichte, erläutert die Theorie der Kontinentalveschiebung und erörtert wie sich das Gesicht der Küsten verändert hat. Er zeigt auf, wie die vielfältigen Ökosystemleistungen der Küsten immer mehr unter Druck geraten und stellt Maßnahmen vor, die in Zukunft notwendig sein werden, um den Bedrohungen durch Klimawandel und Naturkatastrophen Herr zu werden

World Ocean Review 2015 : living with the oceans 5. Coasts - a vital habitat under pressure

The fifth World Ocean Review (WOR) explores the coastal habitat and the diverse expectations upon this habitat. It provides a glimpse into millions of years of history, elucidates the theory of continental drift and discusses the many ways in which coasts have changed. It also illustrates how the diverse ecosystem services rendered by the coasts are being subjected to increasing pressure, and profiles measures that will be necessary in the future to respond effectively to the threats from both climate change and natural disasters

Development of binding and functional assays for the neuropeptide Y Y 2 and Y 4 receptors

For the discovery and pharmacological characterization of new ligands of G-protein coupled receptors, simple, robust and reliable assays are required. This thesis was aimed at the development of new binding and functional assays for the neuropeptide Y (NPY) receptor subtypes hY2, hY4 and rY4. CHO cells were stably transfected with the hY2 receptor gene and used in a flow cytometric binding assay. Binding of unlabeled receptor ligands in competition with cy5-labeled pNPY was determined at equilibrium. Binding of the fluorescent ligand could be visualized by confocal microscopy. A radioligand binding assay was established and the determined binding constants were in good agreement with the ones obtained from the flow cytometric binding assay and with data from literature, respectively. The existence of a large fraction of spare receptors described in the literature was confirmed. The Y2 receptor expressing cells were further stably transfected with the gene encoding the chimeric G-protein Gqi5, allowing the redirection of the signal transduction pathway towards the PLCβ, resulting in a large increase in intracellular calcium concentration after receptor activation. NPY receptor binding properties of the transfected cells remained unaffected. The calcium signal was quantitated in a flow cytometric and a spectrofluorimetric calcium assay. Functional data of agonists as well as antagonists were determined. Additional stable transfection of the cells with the gene encoding for apoaequorin targeted to the mitochondrium converted the calcium mobilization into a luminescence signal. Assay parameters were optimized and an aequorin assay was established in the 96-well format of a luminescence plate reader. Functional data of selected peptides and nonpeptidic compounds were determined and compared with the fluorescence-based calcium assays. The calcium responses could be visualized by means of confocal microscopy and a CCD camera. New fluorescent ligands for the Y4 receptor were synthesized by coupling the fluorescent dyes cy5 and S0586 to the peptide [K4]-hPP. A flow cytometric binding assay was established for the rat Y4 receptor using stably transfected CHO cells. Using a retroviral approach, the hY4 gene was transduced into P388-D1 cells. After enrichment of receptor expressing cells by sorting with the flow cytometer a cell clone with high receptor expression was isolated. Competition binding of known peptide ligands in the presence of the fluorescent ligands was measured by flow cytometry. Furthermore, CHO cells were co-transfected with the hY4 (containing a Kozak sequence for enhanced protein translation), Gqi5 and mtAEQ (aequorin) gene. Stable cell clones were isolated and flow cytometric binding as well as fluorescence- and luminescence-based functional assays were established. It turned out that the peptide GW1229 behaved as a partial agonist in the spectrofluorimetric as well as in the aequorin assay. The calcium signal could be detected by confocal microscopy and by a CCD camera, indicating that the aequorin assay is applicable to HTS-instruments equipped with a CCD camera. The screening of a small compound library revealed a small molecule with micromolar antagonistic activity at the hY4 receptor, which may be a starting point for the search for new Y4 receptor antagonists. The main advantage of the established aequorin assay is the automation of the injection and recording process. Thereby, it is possible to perform more than 400 single calcium assays per day compared to about 40 assays performed with the spectrofluorimetric or flow cytometric calcium assay. Unlike the use of fluorescence dyes, after addition of the cofactor, washing steps are not required. Dye leakage is not an issue of the aequorin assay. The injection speed, which turned out to be an important parameter, is constant, and because the assay volume is very small (200 µl) costs are low and only small amounts of test compounds are required. The fact that the aequorin assay measures an event more distal in the GPCR signal transduction pathway seems not to affect the determined functional data. Taken together, the established flow cytometric binding assays using fluorescence labeled ligands and stably transfected cells are an innovative alternative to radioligand binding assays. The principle of stable co-expression of receptor, chimeric G-protein and mitochondrially targeted aequorin gene for the development of functional fluorescence- and luminescence-based assays has proven to be an efficient approach. This methodology is transferable to other GPCRs, and will facilitate the search for new GPCR ligands as well as their functional characterization

Application of the guanidine – acylguanidine bioisosteric approach to argininamide-type NPY Y2 receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements but compromising drug-likeness of numerous biologically active compounds including ligands of G-protein coupled receptors (GPCRs). As part of a project focusing on the search for guanidine bioisosteres, argininamide-type neuropeptide Y (NPY) Y2 receptor (Y2R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, NG-acylated argininamides were preferably obtained by guanidinylation using tailor-made mono Boc-protected N-acyl-S-methylisothioureas. The compounds were investigated for Y2R antagonism (calcium assays), Y2R affinity and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the NG-substituted (S)-argininamides showed Y2R antagonistic activities and binding affinities comparable to the parent compound, whereas NG-acylated or -carbamoylated analogs containing a terminal amine were superior (Y2R: Ki and KB values in the low nanomolar range). This demonstrates that the basicity of the compounds, although being by 4-5 orders lower than that of guanidines, suffices to form key interactions with acidic amino acids of the Y2R. The acylguanidines bind with high affinity and selectivity to Y2R compared to Y1, Y4, and Y5 receptors. As derivatization of the amino group is tolerated, these compounds are considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y2R. The results support the concept of bioisosteric guanidine-acylguanidine exchange as a broadly applicable approach to retain pharmacological activity regardless of reduced basicity

Determination of affinity and activity of ligands at the human neuropeptide Y Y4 receptor by flow cytometry and aequorin luminescence.

Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity

Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y(2) receptor ligands.

With respect to the discovery and characterization of neuropeptide Y(2) receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY(2) receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq(i5) enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration-response curves of porcine neuropeptide Y in the presence of the Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement