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In zahlreichen, in der Einleitung bereits näher beschriebenen Studien konnte anxiolytische Wirksamkeit des patentierten ätherischen Öls Silexan sowohl im Tiermodell, als auch am Menschen zur Therapie subsyndromaler und generalisierter Angsterkrankungen demonstriert werden.
Das Ziel der vorliegenden Dissertation war es, den zugrunde liegenden pharmakologischen Wirkmechanismus zu untersuchen. Nach eingehender Analyse der Targets klassischer Anxiolytika, richtete sich der Fokus auf spannungsabhängige Calciumkanäle, die die Bindungsstelle des Gabapentinoids Pregabalin enthalten. Die mögliche Modulation der Kanäle wurde an PC-Zellen, einem Modell neuronaler Vorläuferzellen, murinen Synaptosomen, aber auch in primären Neuronen der für die Genese der Angsterkrankungen relevanten Hirnareale Hippocampus und Cortex untersucht. Die Kanalexpression, sowie - distribution in den unterschiedlichen Geweben wurde charakterisiert und die Frage erörtert, ob Silexan spezifische Modulation eines bestimmten Kanalsubtyps vermittelt oder unspezifisch alle Kanäle dieser Gattung inhibiert. Um dies näher zu beleuchten wurden elektrophysiologische Messungen an transfizierten Zellen durchgeführt, die jeweils nur einen Subtyp der Familie spannungsabhängiger Calciumkanäle exprimieren. Zur weiteren Aufklärung des Wirkmechanismus wurde auch die mögliche Involvierung G-Protein gekoppelter Rezeptoren ermittelt.
Der zweite Teil der vorliegenden Untersuchung widmet sich der Klärung potentieller antidepressiver Wirkungen durch mögliche neurotrophe Effekte. Primäre hippocampale Neurone und PC12-Zellen wurden hierzu u.a. auf prä- und postsynaptische Marker, Ausdifferenzierungsmarker, sowie Neuritenwachstum hin analysiert.
Die Proteomforschung wurde die letzten beiden Dekaden maßgeblich durch die Massenspektrometrie geprägt und vorangetrieben. Ohne die Ionisationstechniken MALDI und ESI wäre die Analyse von Peptiden und Proteinen nicht in dem Maße möglich. Durch das Zusammenspiel zwischen Probenvorbereitung und effektiven Trennmethoden mit hochauflösenden Massenspektrometern und Auswertungssoftware können heute problemlos komplexe Proteinmischungen oder ganze Proteome untersucht werden.
Um Proteine in Peptide zu schneiden, wird in den allermeisten Fällen die Protease Trypsin verwendet, deren Eigenschaften in vielerlei Hinsicht die bestmögliche Lösung für die nachfolgende Analyse mit Massenspektrometern bieten. Allerdings stößt die Anwendbarkeit dieses Enzyms bei der Analyse von einigen Proteinen oder Proteinklassen wie Membranproteinen an ihre Grenzen, da nur sehr wenige potentielle Schnittstellen vorhanden sind. In solchen Fällen wurden eine Reihe von weniger spezifischen Enzymen wie Chymotrypsin, Proteinase K oder Elastase in den vergangenen Jahren genutzt, die Proteine auch in für Trypsin weniger gut zugänglichen Bereichen wie Transmembranhelices, in massenspektrometrisch analysierbare Peptide spalten können.
Allerdings stellen die wenig spezifischen Enzyme und die von ihnen generierten Peptide die Massenspektrometrie vor neue Herausforderungen. Für eine Identifizierung benötigen die Peptide eine sehr hohe Massengenauigkeit, daneben sind insbesondere bei der Verwendung von MALDI-Massenspektrometern neutrale und sehr saure Peptide schwerer ionisierbar und analysierbar als basische.
Genügte es bis vor einigen Jahren, nur die Identität einzelner Proteine in komplexen Proben zu bestimmen, hat sich die Fragestellung mittlerweile einem Wandel unterzogen. Heute ist man daran interessiert, wie viel eines bestimmten Proteins vorliegt, besonders im Vergleich mit anderen, unterschiedlich behandelten Proben ist die Regulation von Proteinen von Interesse. Zum Quantifizieren stehen viele unterschiedliche Methoden zur Verfügung. Eine solche stellen die isobaren Derivatisierungsreagenzien TMT und iTRAQ dar, mit denen unterschiedliche Proben nach Peptidfragmentierung quantifiziert werden können.
Fast alle Arbeiten zur Quantifizierung in der Vergangenheit benutzten Trypsin als Protease.
Im Zuge dieser Arbeit sollten die Vorteile, die durch die Verwendung von Elastase bei der Identifizierung von Membranproteinen bereits gezeigt werden konnten, auf die Quantifizierung mit TMT erweitert werden.Wurde in der Vergangenheit noch in manchen Publikationen davon abgeraten, Elastase zu verwenden,weil die Nutzbarkeit der dabei gebildeten komplexen Peptidmischungen in Frage gestellt wurde, konnte in dieser Arbeit gezeigt werden, dass Elastase wie auch Trypsin sich eignen, als Enzym für Quantifizierungsexperimente verwendet zu werden. Dies wurde an Modellproteinen evaluiert und dann auf komplexe Membranproben von Hefezellen erweitert.
Bei Vorexperimenten zur Derivatisierung mit TMT wurde desweiteren festgestellt, dass Peptidklassen, die zuvor nur mit ESI als Ionisationsmethode identifiziert werden konnten, durch die Derivatisierung nun auch mit MALDI zugänglich waren. Die dadurch analysierten kleinen, hydrophoben und sehr sauren Peptide lieferten bei der Kombination mit der underivatisierten Probe einen deutlichen Zugewinn in der Sequenzabdeckung der identifizierten Proteine.
Ein weiterer Teil der Arbeit beschäftigt sich mit der nachträglichen Korrektur von gemessenen Peptidmassen über selbst geschriebene Softwarelösungen für verschiedene Massenspektrometer. Es wurde das Ziel verfolgt, eine möglichst hohe Massengenauigkeit und damit hohe Anzahl an Identifizierungen von Proteinen nach Verdau mit wenig spezifischen Proteasen zu erreichen. Weitere Computerprogramme wurden mit dem Ziel geschrieben, den Arbeitsablauf zu erleichtern und zu verbessern.
Für die früher schon beschriebene Kombination zweier Massenspektrometer mit hoher Massengenauigkeit und Auflösung auf der einen Seite und effizienter Peptidfragmentierung auf der anderen Seite konnte durch Veränderung der Instrumentierung und Software nun eine Automatisierbarkeit geschaffen werden, die es ermöglicht, die Methode standardmäßig bei Routineanalysen zu verwenden.
So ergeben sich viele neue Möglichkeiten neben den oft gewählten Standardprotokollen mit der Analyse tryptischer Verdauansätze mittels LC-ESI-MS/MS, die häufig nur der Einfachheit halber und ohne Anpassung an die eigene Zielsetzung gewählt werden.
Die Arbeit zeigt aber auch auf, dass die Verwendung weniger spezifischer Enzyme sowohl eine Optimierung des Arbeitsablaufs als auch eine Datenauswertung benötigt, die die Besonderheiten der Proteasen berücksichtigt. Wenn dies gewährleistet wird, kann vor allem mit dem Zugewinn durch die Derivatisierung mit TMT eine wertvolle Alternative zu Trypsin genutzt werden.
Conjugated vaccines consisting of flagellin and antigen activate TLR5 and induce strong innate and adaptive immune responses. Objective of the present study was to gain further insight into the mechanisms by which flagellin fusion proteins mediate their immune modulating effects. In a mouse model of Ova-induced intestinal allergy a fusion protein of flagellin and Ova (rflaA:Ova) was used for intranasal and intraperitoneal vaccination. Aggregation status of flaA, Ova and flaA:Ova were compared by light scattering, uptake of fluorescence labeled proteins into mDC was analyzed, processing was investigated by microsomal digestion experiments. Mechanism of DC-activation was investigated using proteasome and inflammasome inhibitors. Immune responses of wildtype, IL-10−/−, TLR5−/− mDCs and Ova-transgenic T cells were investigated. Mucosal and i.p.-application of rflaA:Ova were able to prevent allergic sensitization, suppress disease-related symptoms, prevent body weight loss and reduction in food uptake. Intranasal vaccination resulted in strongest suppression of Ova-specific IgE production. These protective effects were associated with increased aggregation of rflaA:Ova and accompanied by tenfold higher uptake rates into mDC compared to the mixture of both proteins. Microsomal digestion showed that stimulation with rflaA:Ova resulted in faster degradation and the generation of different peptides compared to rOva. rflaA:Ova-mediated activation of mDC could be suppressed in a dose-dependent manner by the application of both inflammasome and proteasome inhibitors. Using TLR5−/− mDC the rflaA:Ova induced IL-10 secretion was shown to be TLR5 dependent. In co-cultures of IL-10−/− mDC with DO11.10 T cells the lack of rflaA:Ova-mediated IL-10 secretion resulted in enhanced levels of both TH2 (IL-4, IL-5) and TH1 (IL-2 and IFN-y) cytokines. In summary, mucosal vaccination with flaA:Ova showed strongest preventive effect. Stimulation with rflaA:Ova results in strong immune modulation mediated by enhanced uptake of the aggregated fusion protein, likely resulting in a different processing by DC as well as stronger TLR5 mediated cell activation.
Lipid mediators have been referred as bioactive lipids, whose change in lipid levels resulted in functional or pathophysiological consequences. They are in the focus of biological research, nevertheless this is a late recognition due to the many difficulties of working with bioactive lipids due to their properties: hydrophobic, unstable and they occur in only in small quantities. Liquid chromatography and mass spectrometry have facilitated the work with them. Especially in this field, cardiovascular diseases and inflammatory mediated diseases and cancer are pathophysiological events where LMs are deregulated. Additionally, if the modulation of one LM pathway is not sufficient to overcome a disease, the combination of targeting two or more pathways could be effective. Needless to say, lipid signaling cascades are complicated pathways and possible shunting into other pathways when inhibiting or genetically deleting enzymes should be taken into consideration.
The first part of this work has focused on enzymes that metabolize eicosanoids, like mPGES-1 and 5-LO. mPGES-1 is an important enzyme metabolizing PGH2 and one of the key players of the AA cascade. Its product, PGE2 plays an important role in different inflammatory processes. Inhibition of the mPGES-1 might be a promising step to circumvent COX dependent side effects of NSAIDs. The class of quinazoline compounds around the lead structure FR20 has been investigated on isolated human and murine enzyme, in HeLa cells and in different human whole blood (HWB) settings to establish the possible effects of these compounds on eicosanoid profiling. Novel compounds with inhibitory activities in the submicromolar range (IC50: 0.13 µM - 0.37 µM on isolated enzyme) were obtained which were also effective in cells and HWB. Furthermore, pharmacological profiling of toxicity and lipid screening with LC/MS-MS revealed that compounds also reduce PGE2 levels in intact cells and whole blood; they do not impair cell viability but lack the ability to inhibit the murine mPGES-1 enzyme. This problem could be overcome by means of chemical synthesis varying the scaffold (quinoline, quinazoline) or introducing biosteric replacement in the phenyl moieties.
5-LO is a relevant enzyme that plays an important role in eicosanoid signaling in particular in leukotriene biosynthesis. Leukotrienes are involved in asthma, allergic rhinitis, glomerulonephritis, rheumatoid arthritis, sepsis, cancer and atherosclerosis. Moreover, genetic variants in the genes of the 5-LO pathway have been associated with the risk of development of acute myocardial infarction and stroke. Eicosanoids are increased in infectious exacerbations of chronic obstructive pulmonary disease (COPD). They are also elevated in the airways of stable COPD patients compared to healthy subjects. Therefore, 5-LO has attired the scientific community as a possible therapeutic target to treat the several disease conditions listed before. In this study an extensive evaluation of imidazo[1,2-a]pyridines as a suitable lead structure for novel 5-LO targeting compounds was presented Within the three publications, 5-LO inhibitory activity of synthesized compounds was investigated in intact PMNL, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and compounds were in vitro pharmacological evaluated. Chemical modifications for lead optimization via straight forward synthesis were used to combine small polar groups (hydroxy, and methoxy groups) which led to a suitable candidate with desired in vitro pharmacokinetic profile in terms of solubility and intrinsic clearance without showing any cytotoxicity. More than 70 imidazo[1,2-a]pyridine derivatives have been synthesized, resulting in more than 50 active compounds. Although it was not possible to introduce a solubility group without impairing the 5-LO inhibitory activity, combination of small polar groups lead to a more favorable solubility and in vitro metabolic stability. Overall, the development of 5-LO inhibitors with high efficacy and selectivity in vivo will provide a possible treatment for patients having one of the diseases where leukotriene biosynthesis plays an important role.
Other types of 5-LO inhibitors have been synthesized during this work, NO-NSAIDs can be postulated as novel 5-LO inhibitors that could circumvent the undesired side-effects of inhibiting COX isoforms (ulcer perforation, gastrointestinal bleeding and in some cases death). It is suggested that NO group is released in situ or after compounds are metabolized. NO-NSAIDs maintain the same anti-inflammatory properties by inhibiting 5-LO in clinical relevant concentrations. NO-NSAIDs are currently under clinical trial for the treatment of diseases where inflammation plays an important role. Synthesis of NO-NSAIDs is straightforward and can be applied for most NSAIDs recently published. Among them, the most promising candidate is NO-sulindac that was able to inhibit 5-LO product formation in intact PMNL, purified 5-LO and HWB in micromolar concentration. Additional experiments regarding their mechanism are currently being performed.
The present study could show that dual inhibitors are an interesting approach that is practicable. It has been used in the recent years to overcome side-effects and diseases concerning more pathophysiological conditions. MetS is an example of a conjunction of symptoms: hyperglycemia, hypertriglyceridemia, hypertension and obesity. Due to its complex nature, the current treatment strategies of MetS require multiple pharmacological compounds regulating lipid and glucose homeostasis as well as blood pressure and coagulation. This study describes the first synthesis of dual sEH/PPAR modulators as potential agents for treatment of MetS. Following a combinatorial approach, an acidic head group known as a pharmacophore important for PPARα/γ dual agonistic activity was combined with different hydrophobic urea derivatives in order to introduce an epoxide mimetic (sEH pharmacophore). The resulting compounds yielded high inhibition of sEH and different patterns of PPAR agonistic activity. This study demonstrates that the pharmacophores of PPAR agonists and sEH inhibitors can be easily combined, resulting in a simplified blueprint of a dual sEH/PPAR modulator. Further in vivo pharmacological evaluation studies are needed in order to evaluate, which pattern of PPAR activation shows the most promising profile for treatment of metabolic syndrome.
Another example of dual pharmacology has been presented in this work. Natural products derived compounds were able to target sEH and exhibit promising antiproliferative properties. The principle of addressing multiple targets by natural products can be transferred to synthetic multi-target ligands. In conclusion, several (E)-styryl-1H-benzo[d]imidazoles were synthesized and evaluated on recombinant sEH after an initial hit (IPS) that lead to potent sEH inhibitors exhibiting antiproliferative activities. Following the natural product-inspired design, the desired biological activity from a bacterial secondary metabolite has been enhanced and transferred to a synthetic compound series. The resulting compounds were accessible via an easy synthetic route and offered a possibility to investigate the structure-activity relationships. The natural product inspired drug design extends the valuable role of natural products as drugs and drug precursors to templates for fully synthetic bioactive molecules. Simplification of natural products by means of chemical synthesis could lead to an interesting field in the treatment of cancer.
Affinity chromatography has been used to unravel unknown- and off-target effects which either contribute to the biological effect of the inhibitor or that counteract or lead to undesired side-effects. During this PhD work, two main projects related to this technique have been established. In the first one, related to an imidazo[1,2-a]pyridine inhibitor (EP6), it has been shown that epoxide-sepharose is a reliable material in order to couple compounds bearing an alcohol. Coupling of an analogue of EP6 to the sepharose has been accomplished and affinity towards 5-LO was demonstrated. The challenging step is to discern from unspecific protein binders and analysis via SDS-PAGE separation and mass spectrometry. Further experiments using other cell types or improving SDS-PAGE analysis (e.g. 2D gel analysis) should be useful to unravel EP6 off-target effect. During the second project related to off-target effects of celecoxib and DMC, the main problem was the coupling of the functional group to the sepharose. Affinity towards COX-2 could not be demonstrated pointing out the inefficient coupling method. Higher pH values during coupling reaction should be tested in further experiments. Nevertheless, affinity chromatography is a useful technique to unravel cellular mechanisms.
Sphingolipid metabolism is also a recent area that attired the attention of cancer researchers, due to their important roles in cell proliferation and apoptosis. Ceramide metabolism inhibitors were synthesized and evaluated on different assay systems in order to assess their efficacy on several cancer lines. Remarkably, 2,2-dimethyl-1,3-dioxolan-4-yl)methanamine (32) was a useful scaffold to mimic the sphingoid base. This key intermediate was used to produce ceramide analogues that could enter the cell and target apoptosis machinery. EB143 (38) increased ceramide levels in an in vitro ceramide synthase assay in a dose-response manner meaning that ceramide synthase was not inhibited but the ceramide de novo synthesis was activated. This effect was due to the fact that EB143 is a cytotoxic compound with an interesting antiproliferative profile. Further chemical modifications should be carried out to modulate this effect.
COX and LO inhibitors are cancer-preventive not only by inhibiting specific antiapoptotic AA metabolites but also by facilitating accumulation of AA which promotes neutral SMase activity and increases the proapoptotic ceramide. Several 5-LO inhibitors have been evaluated on several cancer lines and sphingolipid levels were measured in order to obtain a relationship. A549, Capan-2 and MCF-7 cells line were incubated with synthetic 5-LO inhibitors and zileuton. Compounds were cytotoxic to all cancer cell lines except from A549. Needless to say, zileuton did not exhibit a cytotoxic profile. Synthetic 5-LO inhibitors were able to modify ceramide levels but were useless when coincubating with sphingolipid metabolism inhibitors (myoricin, amitryptiline etc.) and inconsistent results were obtained. On the contrary, zileuton selectively increased Cer-C16 levels and in less extend Cer-C24:1. When using a SPT inhibitor (myoricin) alone was able to reduce C24:1 and Cer-C16:0 levels below the control, a similar effect occurred when incubation the cells with zileuton and myriocin. Interestingly, treatment of zileuton together with either amitryptiline or desipramine led to a decrease in Cer-C24:1 and levels Cer-C16:0 but the inhibition was not complete indicating that probably the de novo pathway has an important role. Further investigations on mRNA level should be carried out in order to discern which CerS is activated.
The main objective of the present thesis was the synthesis of lipid signaling modulators and their evaluation in vitro as therapeutic strategy to overcome pathophysiological conditions (cancer, metabolic syndrome, etc). It has been accomplished on many relevant targets like 5-LO, mPGES-1, sEH and PPAR and these lipid signaling modulators could be used in the treatment of diseases conditions where lipid mediators play an important role.