The systematic annotation of the three main GPCR families in Reactome

Reactome is an open-source, freely available database of human biological pathways and processes. A major goal of our work is to provide an integrated view of cellular signalling processes that spans from ligand–receptor interactions to molecular readouts at the level of metabolic and transcriptional events. To this end, we have built the first catalogue of all human G protein-coupled receptors (GPCRs) known to bind endogenous or natural ligands. The UniProt database has records for 797 proteins classified as GPCRs and sorted into families A/1, B/2 and C/3 on the basis of amino accid sequence. To these records we have added details from the IUPHAR database and our own manual curation of relevant literature to create reactions in which 563 GPCRs bind ligands and also interact with specific G-proteins to initiate signalling cascades. We believe the remaining 234 GPCRs are true orphans. The Reactome GPCR pathway can be viewed as a detailed interactive diagram and can be exported in many forms. It provides a template for the orthology-based inference of GPCR reactions for diverse model organism species, and can be overlaid with protein–protein interaction and gene expression datasets to facilitate overrepresentation studies and other forms of pathway analysis

Reactome: a database of reactions, pathways and biological processes

Reactome (http://www.reactome.org) is a collaboration among groups at the Ontario Institute for Cancer Research, Cold Spring Harbor Laboratory, New York University School of Medicine and The European Bioinformatics Institute, to develop an open source curated bioinformatics database of human pathways and reactions. Recently, we developed a new web site with improved tools for pathway browsing and data analysis. The Pathway Browser is an Systems Biology Graphical Notation (SBGN)-based visualization system that supports zooming, scrolling and event highlighting. It exploits PSIQUIC web services to overlay our curated pathways with molecular interaction data from the Reactome Functional Interaction Network and external interaction databases such as IntAct, BioGRID, ChEMBL, iRefIndex, MINT and STRING. Our Pathway and Expression Analysis tools enable ID mapping, pathway assignment and overrepresentation analysis of user-supplied data sets. To support pathway annotation and analysis in other species, we continue to make orthology-based inferences of pathways in non-human species, applying Ensembl Compara to identify orthologs of curated human proteins in each of 20 other species. The resulting inferred pathway sets can be browsed and analyzed with our Species Comparison tool. Collaborations are also underway to create manually curated data sets on the Reactome framework for chicken, Drosophila and rice

The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/similar to ismel/GPCR-Nets/index.html

The G protein-coupled receptor heterodimer network (GPCR-HetNet) and its hub components

G protein-coupled receptors (GPCRs) oligomerization has emerged as a vital characteristic of receptor structure. Substantial experimental evidence supports the existence of GPCR-GPCR interactions in a coordinated and cooperative manner. However, despite the current development of experimental techniques for large-scale detection of GPCR heteromers, in order to understand their connectivity it is necessary to develop novel tools to study the global heteroreceptor networks. To provide insight into the overall topology of the GPCR heteromers and identify key players, a collective interaction network was constructed. Experimental interaction data for each of the individual human GPCR protomers was obtained manually from the STRING and SCOPUS databases. The interaction data were used to build and analyze the network using Cytoscape software. The network was treated as undirected throughout the study. It is comprised of 156 nodes, 260 edges and has a scale-free topology. Connectivity analysis reveals a significant dominance of intrafamily versus interfamily connections. Most of the receptors within the network are linked to each other by a small number of edges. DRD2, OPRM, ADRB2, AA2AR, AA1R, OPRK, OPRD and GHSR are identified as hubs. In a network representation 10 modules/clusters also appear as a highly interconnected group of nodes. Information on this GPCR network can improve our understanding of molecular integration. GPCR-HetNet has been implemented in Java and is freely available at http://www.iiia.csic.es/~ismel/GPCR-Nets/index.html. © 2014 by the authors; licensee MDPI, Basel, Switzerland.This work has been supported by the Swedish Royal Academy of Sciences (Stiftelsen B. von Beskows Fond and Stiftelsen Hierta-Retzius stipendiefond) and Karolinska Institutets Forskningsstiftelser 2011 and 2012 to D.O.B.-E., by grants from the Swedish Medical Research Council (04X-715), Telethon TV3’s La Marató Foundation 2008 and Hjärnfonden to K.F., D.O.B.-E., I.B. and W.R.-F. belong to the “Academia de Biólogos Cubanos” group. Feliciano Calvo and Carmelo Million are acknowledged for their support during the GPCR heterodimer list preparation.Peer Reviewe

Adult Hippocampal Neurogenesis Can Be Enhanced by Cold Challenge Independently From Beigeing Effects

In this study, we investigated the effects of cold challenge on adult hippocampal neurogenesis (AHN) and hippocampal gene expression and whether these are mediated by beigeing of peripheral fat tissues. Cold challenge (6 ± 2°C) for 1 and 4 weeks was found to induce beigeing effects in inguinal white adipose tissue based on hematoxylin and eosin staining as well as uncoupled protein-1 immunohistochemical staining. In the hippocampus, cold challenge for 1 or 4 weeks increased dentate gyrus neurogenesis and expression of genes related to AHN, including notch signaling, G protein-coupled receptor signaling, and adrenergic beta receptor-1. However, this enhancement of neurogenesis and gene expression by cold challenge was not shown by administration of CL 316,243, which induces peripheral beigeing similar to cold challenge but does not cross the blood–brain barrier. These results suggest that cold challenge promotes AHN and central expression of AHN-related, signaling, and β1-adrenergic receptors genes, and that peripheral beigeing by itself is not sufficient to mediate these effects. Considering the increase in AHN and gene expression changes, cold challenge may offer a novel approach to hippocampal modulation

In vivo pharmacological profiling in Xenopus embryos defines a subset of A1 adenosine receptor-selective antagonists with potent anti-angiogenic activities

The purine nucleoside adenosine is an important intermediate metabolite, which is released from most cells. It mediates signaling through activation of four distinct adenosine receptors (ARs): A1, A2A, A2B, and A3. ARs have been recognized as therapeutic targets for a wide range of physiological processes and pathophysiological conditions, including epilepsy, chronic pain, ischemia, inflammation, and cancer. This has led to the development of various subtype-specific AR antagonists. By screening a chemical library in vivo, we had previously recovered AR antagonists as inhibitors of blood and lymph vessel angiogenesis in Xenopus embryos [Kalin et al. 2009 Blood 114, 1110-1122]. Here, we assessed the role of adenosine signaling in vascular development in Xenopus embryos by testing a panel of subclass-selective AR antagonists for anti-(lymph) angiogenic activities. Compound-treated embryos were assessed phenotypically for evidence of edema formation, which is a reliable pathophysiological indicator of anti-angiogenic activity. Quantitative pharmacological parameters, such as EC50, ECmax, and LC50 values, were established for each compound. While antagonists selective for A2A, A2B, or A3 ARs were typically inactive, all A1 AR antagonists were found to induce edema with EC50 values ranging from 0.3 to 6.7 µM. However, only two compounds, 7-chloro-4-hydroxy-2-phenyl-1, 8-naphthyridine (7CN) and 1,3 Diethyl-8-phenylxanthine (DEPX), had potent anti-angiogenic activities in vivo. Subsequently, structure-activity relationship studies led to the identification of two additional compounds, 7-methyl-2-phenyl-1, 8-naphthyridine (7MN) and 1,3-dipropyl-8-phenylxanthine (DPPX), equally capable of disrupting vascular development in Xenopus embryos. Treatment of primary human endothelial cell cultures demonstrated that only 7CN and 7MN but not DEPX and DPPX disrupted cell proliferation and VEGF-induced sprouting in vitro. Gene expression and knockdown studies in Xenopus embryos failed however to provide evidence for a central role of A1 ARs in vascular development. This suggests that the antiangiogenic activities of 7CN and 7MN may be due to polypharmacology involving inhibition of other targets than A1 ARs alone. In fact, we found that 7CN and 7MN induced destabilization of microtubules, mitotic arrest, and excess DNA replication in endothelial cell cultures. Taken together, phenotypic compound profiling in Xenopus embryos has led to the identification of 7CN and 7MN as promising drug candidates that act as potent anti-angiogenic compounds interfering with essential endothelial cell functions.Das Purinnukleosid Adenosin ist ein wichtiger Metabolit, der von vielen Zellen freigesetzt wird. Adenosin wirkt als Signalmolekül mittels Aktivierung von vier verschiedenen Adenosinrezeptoren (AR): A1, A2A, A2B und A3. AR dienen als vielversprechende Angriffspunkte für die Entwicklung von Arzneimitteln zur Modulation einer breiten Palette von physiologischen Prozessen und pathophysiologischen Zuständen, einschliesslich Epilepsie, chronischen Schmerzen, Ischämie, Entzündungen und Krebs. In der Folge wurden verschiedene subtypspezifische AR-Antagonisten entwickelt. Mittels eines In Vivo Screening-Verfahrens hatte unsere Arbeitsgruppe zuvor mehrere AR-Antagonisten einer chemischen Bibliothek als Inhibitoren der Blut- und Lymphgefässangiogenese in Xenopus Embryonen identifziert [Kalin et al. 2009 Blood 114, 1110-1122]. In der vorliegenden Arbeit untersuchten wir mittels einer Reihe von subklassenselektiven AR-Antagonisten die Rolle der Signalwirkung von Adenosin bei der Bildung des Gefässsystems in Xenopus Embryonen. Insbesondere wurde nach anti-lymphatischen bzw. -angiogenetischen Aktivitäten in vivo gesucht. Wirkstoff-behandelte Embryonen wurden phänotypisch beurteilt und auf Ödembildung als zuverlässigen pathophysiologischen Indikator für anti-angiogenetische Aktivitäten untersucht. Für jede Verbindung wurden quantitative pharmakologische Parameter, wie EC50, ECmax und LC50, ermittelt. Typischerweise waren A2A-, A2B- oder A3-AR-selektive Antagonisten inaktiv. Hingegen waren alle A1 AR-Antagonisten aktiv und induzierten Ödeme mit EC50-Werten von 0.3 bis 6.7 µM. Allerdings besassen nur zwei Verbindungen, 7-Chloro-4-hydroxy-2-phenyl-1, 8-naphthyridin (7CN) und 1,3 Diethyl-8-phenylxanthin (DEPX), starke anti-angiogenetische Aktivitäten in vivo. Anschliessend wurden Studien zur Struktur-Wirkungs-Beziehung durchgeführt. Diese führten zur Identifizierung von zwei zusätzlichen Verbindungen, 7-Methyl-2-phenyl-1, 8-naphthyridin (7MN) und 1,3-Dipropyl-8-phenylxanthin (DPPX), die in der Lage waren, die Gefässbildung in Xenopus Embryonen zu stören. Die Behandlung von primären humanen Endothelzellkulturen zeigte dann, dass nur 7CN und 7MN, aber nicht DEPX und DPPX, in der Lage waren die Proliferation und das VEGF-induzierte Sprouting in vitro zu unterbinden. Umfangreiche Genexpressions- und Knockdown-Studien lieferten keine Hinweise für eine zentrale Rolle von A1 AR bei der vaskulären Gefässbildung in Xenopus Embryonen. Dies weist darauf hin, dass die anti-angiogenetischen Aktivitäten von 7CN und 7MN vermutlich auf Polypharmakologie zurückzuführen sind und sich nicht durch die alleinige Hemmung von A1 ARs erklären lassen. In der Tat fanden wir, dass die Behandlung von endothelialen Zellkulturen mit 7CN und 7MN zu einer Destabilisierung des mikrotubulären Zytoskeletts, einer Blockierung der Mitose und zu übermässiger DNA Replikation führten. Zusammengenommen hat die phänotypische Charakterisierung von AR Antagonisten in Xenopus Embryonen zur Identifizierung von 7CN und 7MN als viel versprechende Wirkstoffkandidaten geführt. Beide Verbindugen haben potente anti-angiogenetische Aktivitäten, welche wesentliche endotheliale Zellfunktionen stören

Modulation of Network Oscillations by Brain Stimulation

Finding new and effective treatments for mental illness represents one of the largest challenges of our time due the large number of people affected. Despite long and careful study there have been few recent breakthroughs in pharmacological treatments of mental illness. To address this, the National Institute of Mental Health (NIMH) has recently begun to focus on the investigation of network level correlates of mental illnesses. Patients with mental illness often exhibit aberrant neural oscillatory activity, thus making the network level a promising scale for the identification of measureable neural correlates of mental illnesses. At the network level, neural activity is primarily in the form of cortical oscillations which may be recorded noninvasively with electroencephalography (EEG). Such EEG oscillations are the result of synchronized activity from many cells in the neocortex. However the exact mechanisms of how oscillations arise and spread throughout the brain remain unknown. Non-invasive brain stimulation is a promising treatment modality because it specifically targets activity of brain networks. Unlike pharmacological treatments, stimulation with electric and magnetic fields directly targets electrical activity of many cells in a network. In particular, transcranial alternating current stimulation appears to be especially suited for targeting oscillations in brain networks. Despite the promise of these brain stimulation techniques, the underlying mechanisms remain unknown. The studies presented in this dissertation address two critical gaps in the treatment of mental illnesses. (1) How does rhythmic network activity arise from cellular and synaptic components? And (2) how does brain stimulation interact with ongoing network activity? Only by understanding how network activity arises and how it interacts with brain stimulation we may begin to design brain stimulation paradigms for treatment of mental illness.Doctor of Philosoph

Potenzial der organotypischen Kultur der Haut sowie des Hauttumors als ein in-vivo-ähnliches Untersuchungsinstrument

Einleitung: Organotypische Kulturen der Haut und des Plattenepithelkarzinoms (PEK) eröffnen neue Möglichkeiten, spezifische Fragestellungen unter in-vivo-ähnlichen Bedingungen zu analysieren. In der vorliegenden Arbeit wurde das Potenzial der organotypischen Kultur als Untersuchungsinstrument für die Haut und für Hauttumoren evaluiert, indem der Einfluss von Mastzellen, Histamin und des Angiotensin-II-Rezeptor-Typ-2-Agonisten Compound 21 (C21) auf Keratinozyten und PEK-Zellen in der organotypischen Kultur analysiert wurden. Methode: Für die Untersuchung wurden Keratinozyten, Fibroblasten und Mastzellen aus Hautgewebe isoliert. Als PEK-Zellen wurden die Zelllinien SCC12 und SCC13 analysiert. Die organotypische Kultur bestand aus drei Komponenten: einer azellulären Schicht aus Kollagen, einem Dermisäquivalent, das Fibroblasten und Mastzellen beinhaltete, und einer epithelisierten Schicht mit Keratinozyten bzw. SCC12- oder SCC13-Zellen. 6–8 Tage nach dem Air-Lifting wurde die Kultur histologisch bearbeitet. Ergebnisse: Die organotypischen Kulturen der Haut wurden sowohl mit Keratinozyten als auch mit PEK-Zellen mit und ohne Integration von Mastzellen erfolgreich etabliert. Dabei zeigten die Keratinozyten und PEK-Zellen in der organotypischen Kultur Wachstumsmuster, welche in der Monokultur nicht zu erkennen waren. Durch die Anwesenheit oder Aktivierung der Mastzellen ergaben sich keine Veränderungen bei den PEK-Zellen. Allerdings kam es nach Histamin-Gabe zu einer signifikant reduzierten Proliferation von SCC12- und SCC13-Zellen mit einer verminderten Expression des Vascular endothelial growth factor receptor 2 (VEGFR2). C21 führte in beiden Zelllinien zu einer Reduktion der Proliferation sowie zu einer Induktion der Apoptose. Diese anti-proliferativen Effekte von Histamin und C21 traten in der Monokultur nicht auf. C21 wirkte auch anti-proliferativ und pro-apoptotisch auf Keratinozyten in der organotypischen Kultur. Diskussion: In der vorliegenden Arbeit gelang es erstmals, erfolgreich eine organotypische Kultur der Haut mit PEK- und Mastzellen zu etablieren. Die vielfältigen Einsatzmöglichkeiten dieser organotypischen Kultur konnten repräsentativ durch die Analyse des Effekts von Mastzellen, Histamin und C21 demonstriert werden. PEK-Zellen verhielten sich in der organotypischen Kultur ‚in-vivo-ähnlicher’ als in einer Monokultur, was die Überlegenheit der organotypischen Kultur belegt. Die proliferationshemmende Wirkung von Histamin mit einer verminderten Expression von VEGFR2 erklärt die tumorprotektive Rolle von Mastzellen. Daher sollten VEGFR2-Antagonisten zur therapeutischen Anwendung weiterhin getestet werden. Die anti-proliferative und pro-apoptotische Wirkung von C21 auf PEK-Zellen und Keratinozyten in der organotypischen Kultur deutet auf ein therapeutisches Potenzial dieser Substanz bei Erkrankungen wie PEK oder Psoriasis hin.Introduction: The organotypic culture of skin and squamous cell carcinoma (SCC) opens up a new opportunity to analyze specific questions under in vivo-like conditions. In the present work, the potential of organotypic culture as an investigational tool for the skin and cutaneous tumors was evaluated by analyzing the effects of mast cells and histamine as well as angiotensin II type 2 receptor agonist Compound 21 (C21) on keratinocytes and SCC cells in the organotypic culture. Method: Keratinocytes, fibroblasts and mast cells were isolated from the skin tissue for investigation. Two cell lines SCC12 and SCC13 were analyzed as cells of SCC. The organotypic culture consists of three components: an acellular layer of collagen, a dermal equivalent containing fibroblasts and mast cells, and an epithelialized layer of keratinocytes and SCC12 or SCC13, respectively. In 6 to 8 days after the air-lifting, the culture was histologically processed. Results: The organotypic cultures of the skin with keratinocytes as well as with SCC cells with and without integration of the mast cells were successfully established. The keratinocytes and SCC cells showed growth patterns in the organotypic culture, which were not detected in the monoculture. The presence or activation of the mast cells did not result in any changes in the SCC cells. However, after administration of histamine, there was a significantly reduced proliferation of SCC12 and SCC13 with an underexpression of vascular endothelial growth factor receptor 2 (VEGFR2). C21 led to a reduction in proliferation and an induction of apoptosis in the two cell lines. These antiproliferative effects of histamine and C21 were not demonstrated in the monoculture. C21 also had an antiproliferative and apoptotic effect on keratinocytes in the organotypic culture. Discussion: It was possible for the first time to successfully establish an organotypic culture of the skin with SCC cells and mast cells. The diverse uses of this organotypical culture could be representatively demonstrated by analyzing the effect of mast cells, histamine and C21. SCC cells behaved more "in vivo-like" in the organotypic culture than in a monoculture, which demonstrates the superiority of the organotypic culture. The proliferation-inhibiting effect of histamine with a reduced expression of VEGFR2 explains the tumor-protective role of mast cells. Therefore, VEGFR2 antagonists should be tested for therapeutic use. The anti-proliferative and pro-apoptotic effects of C21 on SCC cells and keratinocytes in organotypic culture indicate a therapeutic potential of this substance in diseases such as SCC or psoriasis

Synthesis and pharmacological characterization of homo- and hetero-dimeric compounds, targeting the hH₁R and/or hH₄R

G-protein coupled receptors are the most important class of biological targets for drug development. Among them, the histamine receptors may be considered as representative examples for aminergic GPCRs. The latest member of the histamine receptor family, the H4R, was reported to be involved in immunological processes and inflammatory diseases. However, the (patho)physiological role of the H4R is far from being fully understood. Thus, potent and selective pharmacological tools in various forms targeting H4R are required. Based on complementary and overlapping functions of hH1R and hH4R, it is presumed that combined H1R/H4R antagonism might be superior to monotherapy in the treatment of allergic diseases. In the first part of this thesis, twenty-two novel homo-dimeric ligands based on the prominent H1R antagonists diphenhydramine (3.1), pyrilamine (3.2) and dual H1R/H4R antagonist quinazoline derivative (3.3), were synthesized to probe putative accessory binding sites on hH1R and hH4R. Furthermore, their binding affinities were determined at the hH1R and/or hH4R. Between the two types of spacers which were employed in this study, connecting chains comprising amide groups exhibited similar affinity compared to their monomeric counterparts. By contrast, alkyl chains without amides showed decreased affinity at the hH1R, suggesting that the lipophilicity of the compounds plays an important role in binding affinity, e.g., high lipophilicity may result a poor solubility and high non-specific binding of the compound in assays. Since the variation of the spacer length had no significant influence on binding affinities, it may be suggested that one pharmacophore of the homo-dimeric ligand was not binding to the hH1R, thus it did not contribute to receptor-ligand binding. At the hH4R, all bivalent quinazoline-type ligands showed no obvious activity. This may be interpreted that the quinazoline-type homo-dimeric ligands are not tolerated at the hH4R. In a word, the data of the present study were not sufficient to prove the existence of accessory binding sites on hH1R and hH4R. The second part of this thesis was focused on developing dual hH1R/hH4R antagonists. Thirty benzimidazole- and quinazoline-type compounds were synthesized and pharmacologically characterized at the four human histamine receptor subtypes. The incorporation of an imidazole moiety, separated from the benzimidazole moiety by an appropriate linker, largely improved the binding affinities at the hH4R and resulted in a balanced dual hH1R/hH4R antagonist (compound 4.35b) with Ki values in the two-digit nM range. However, ligands comprising imidazolylalkyl moieties did not discriminate between hH3R and hH4R. In summary, although the dimeric approach may be an interesting and useful strategy in developing bitopic and dual target ligands targeting H1R and/or H4R, there are numerous problems associated with this approach. Due to the low homology between ligand binding sites of H1R and H4R, it appears extremely difficult to identify a common H1R/H4R pharmacophore at a high level of affinity. Moreover, as the H4R has a relatively high homology with the H3R, a poor discrimination between both receptor subtypes may result in more complex binding and functional properties of hybrid compounds targeting the H4R

In vivo pharmacological profiling in Xenopus embryos defines a subset of A1 adenosine receptor-selective antagonists with potent anti-angiogenic activities

The purine nucleoside adenosine is an important intermediate metabolite, which is released from most cells. It mediates signaling through activation of four distinct adenosine receptors (ARs): A1, A2A, A2B, and A3. ARs have been recognized as therapeutic targets for a wide range of physiological processes and pathophysiological conditions, including epilepsy, chronic pain, ischemia, inflammation, and cancer. This has led to the development of various subtype-specific AR antagonists. By screening a chemical library in vivo, we had previously recovered AR antagonists as inhibitors of blood and lymph vessel angiogenesis in Xenopus embryos [Kalin et al. 2009 Blood 114, 1110-1122]. Here, we assessed the role of adenosine signaling in vascular development in Xenopus embryos by testing a panel of subclass-selective AR antagonists for anti-(lymph) angiogenic activities. Compound-treated embryos were assessed phenotypically for evidence of edema formation, which is a reliable pathophysiological indicator of anti-angiogenic activity. Quantitative pharmacological parameters, such as EC50, ECmax, and LC50 values, were established for each compound. While antagonists selective for A2A, A2B, or A3 ARs were typically inactive, all A1 AR antagonists were found to induce edema with EC50 values ranging from 0.3 to 6.7 µM. However, only two compounds, 7-chloro-4-hydroxy-2-phenyl-1, 8-naphthyridine (7CN) and 1,3 Diethyl-8-phenylxanthine (DEPX), had potent anti-angiogenic activities in vivo. Subsequently, structure-activity relationship studies led to the identification of two additional compounds, 7-methyl-2-phenyl-1, 8-naphthyridine (7MN) and 1,3-dipropyl-8-phenylxanthine (DPPX), equally capable of disrupting vascular development in Xenopus embryos. Treatment of primary human endothelial cell cultures demonstrated that only 7CN and 7MN but not DEPX and DPPX disrupted cell proliferation and VEGF-induced sprouting in vitro. Gene expression and knockdown studies in Xenopus embryos failed however to provide evidence for a central role of A1 ARs in vascular development. This suggests that the antiangiogenic activities of 7CN and 7MN may be due to polypharmacology involving inhibition of other targets than A1 ARs alone. In fact, we found that 7CN and 7MN induced destabilization of microtubules, mitotic arrest, and excess DNA replication in endothelial cell cultures. Taken together, phenotypic compound profiling in Xenopus embryos has led to the identification of 7CN and 7MN as promising drug candidates that act as potent anti-angiogenic compounds interfering with essential endothelial cell functions.Das Purinnukleosid Adenosin ist ein wichtiger Metabolit, der von vielen Zellen freigesetzt wird. Adenosin wirkt als Signalmolekül mittels Aktivierung von vier verschiedenen Adenosinrezeptoren (AR): A1, A2A, A2B und A3. AR dienen als vielversprechende Angriffspunkte für die Entwicklung von Arzneimitteln zur Modulation einer breiten Palette von physiologischen Prozessen und pathophysiologischen Zuständen, einschliesslich Epilepsie, chronischen Schmerzen, Ischämie, Entzündungen und Krebs. In der Folge wurden verschiedene subtypspezifische AR-Antagonisten entwickelt. Mittels eines In Vivo Screening-Verfahrens hatte unsere Arbeitsgruppe zuvor mehrere AR-Antagonisten einer chemischen Bibliothek als Inhibitoren der Blut- und Lymphgefässangiogenese in Xenopus Embryonen identifziert [Kalin et al. 2009 Blood 114, 1110-1122]. In der vorliegenden Arbeit untersuchten wir mittels einer Reihe von subklassenselektiven AR-Antagonisten die Rolle der Signalwirkung von Adenosin bei der Bildung des Gefässsystems in Xenopus Embryonen. Insbesondere wurde nach anti-lymphatischen bzw. -angiogenetischen Aktivitäten in vivo gesucht. Wirkstoff-behandelte Embryonen wurden phänotypisch beurteilt und auf Ödembildung als zuverlässigen pathophysiologischen Indikator für anti-angiogenetische Aktivitäten untersucht. Für jede Verbindung wurden quantitative pharmakologische Parameter, wie EC50, ECmax und LC50, ermittelt. Typischerweise waren A2A-, A2B- oder A3-AR-selektive Antagonisten inaktiv. Hingegen waren alle A1 AR-Antagonisten aktiv und induzierten Ödeme mit EC50-Werten von 0.3 bis 6.7 µM. Allerdings besassen nur zwei Verbindungen, 7-Chloro-4-hydroxy-2-phenyl-1, 8-naphthyridin (7CN) und 1,3 Diethyl-8-phenylxanthin (DEPX), starke anti-angiogenetische Aktivitäten in vivo. Anschliessend wurden Studien zur Struktur-Wirkungs-Beziehung durchgeführt. Diese führten zur Identifizierung von zwei zusätzlichen Verbindungen, 7-Methyl-2-phenyl-1, 8-naphthyridin (7MN) und 1,3-Dipropyl-8-phenylxanthin (DPPX), die in der Lage waren, die Gefässbildung in Xenopus Embryonen zu stören. Die Behandlung von primären humanen Endothelzellkulturen zeigte dann, dass nur 7CN und 7MN, aber nicht DEPX und DPPX, in der Lage waren die Proliferation und das VEGF-induzierte Sprouting in vitro zu unterbinden. Umfangreiche Genexpressions- und Knockdown-Studien lieferten keine Hinweise für eine zentrale Rolle von A1 AR bei der vaskulären Gefässbildung in Xenopus Embryonen. Dies weist darauf hin, dass die anti-angiogenetischen Aktivitäten von 7CN und 7MN vermutlich auf Polypharmakologie zurückzuführen sind und sich nicht durch die alleinige Hemmung von A1 ARs erklären lassen. In der Tat fanden wir, dass die Behandlung von endothelialen Zellkulturen mit 7CN und 7MN zu einer Destabilisierung des mikrotubulären Zytoskeletts, einer Blockierung der Mitose und zu übermässiger DNA Replikation führten. Zusammengenommen hat die phänotypische Charakterisierung von AR Antagonisten in Xenopus Embryonen zur Identifizierung von 7CN und 7MN als viel versprechende Wirkstoffkandidaten geführt. Beide Verbindugen haben potente anti-angiogenetische Aktivitäten, welche wesentliche endotheliale Zellfunktionen stören