Universitätsbibliothek

Alzheimer′s disease (AD) is a neurological disorder with still no preventive or curative treatment. Flavonoids are phytochemicals with potential therapeutic value. Previous studies described the flavanone sterubin isolated from the Californian plant Eriodictyon californicum as a potent neuroprotectant in several in vitro assays. Herein, the resolution of synthetic racemic sterubin (1) into its two enantiomers, (R)‐1 and (S)‐1, is described, which has been performed on a chiral chromatographic phase, and their stereochemical assignment online by HPLC‐ECD coupling. (R)‐1 and (S)‐1 showed comparable neuroprotection in vitro with no significant differences. While the pure stereoisomers were configurationally stable in methanol, fast racemization was observed in the presence of culture medium. We also established the occurrence of extracted sterubin as its pure (S)‐enantiomer. Moreover, the activity of sterubin (1) was investigated for the first time in vivo, in an AD mouse model. Sterubin (1) showed a significant positive impact on short‐ and long‐term memory at low dosages.

Cell culture models are helpful tools to study inflammatory diseases, like rheumatoid arthritis (RA), osteoarthritis (OA), arteriosclerosis or asthma, which are linked to increased matrix metalloproteinase (MMP) activity. Such cell culture models often focus on the secretion of cytokines and growth factors or the direct effects of disease on tissue destruction. Even though the crucial role of MMPs in inflammatory diseases is known, the results of MMP studies are contradictious and the use of MMPs as biomarkers is inconsistent. MMPs play an important role in disease pathology, as they are involved in elastin degradation in the walls of alveoli in chronic obstructive pulmonary disease (COPD), tumor angiogenesis and metastasis and in cartilage and bone degradation in arthropathies. In RA and OA MMPs are secreted by osteocytes, synoviocytes, and by infiltrating immune cells in response to the increased concentration of inflammatory mediators, like growth factors and cytokines. MMPs are zinc and calcium-dependent proteinases and play an important role in physiological and pathological extracellular matrix (ECM) turn over. Their substrate specificity gives them the ability to degrade all major ECM components, like aggrecan, elastin, gelatin, fibronectin and all types of collagen even the triple helix of collagen monomers. The ECM consists of two large three-dimensional cross-linked macromolecule classes: one are fibrous proteins, like collagen and elastin fibers that are responsible for ECM’s structure, tensile strength, resiliency, reversible extensibility, and deformability and the second class is comprised of proteoglycans composed of glycosaminoglycan (GAG) chains covalently attached to protein cores that are multifunctionally involved in signaling pathways and cell interactions. ECM is present within all tissues and organs and changes in ECM structure contribute to pathogenesis, e.g. wounded and fibrotic tissue, COPD or tumours. This thesis primarily focuses on the development of a diagnostic peptide system, that enables to gain information on MMP activity from ECM by deploying the isobaric mass encoding strategy. The core element of the developed system is an isotopically labelled peptide sequence (mass tag), that is released in response to elevated levels of MMPs and allows multiplexed detection in tandem mass spectrometry (LC-MS/MS). The mass reporters possess a modular structure with different functionalities. C-terminal either a transglutaminase (TG) recognition sequence or a high molecular weight polyethylene glycol (PEG) moiety was attached to immobilize the mass reporters covalently or physically at the injection site. The following matrix metalloproteinase substrate sequence (MSS) is incorporated in two different versions with different sensitivity to MMPs. The MSS were applied in pairs for relative quantification consisting of the cleavable version synthesized with natural L-amino acids and the non-cleavable D-amino acid variant. The mass tag was synthesized with isotopically labelled amino acids and is separated from the MSS by a UV light-sensitive molecule. N-terminal the mass tag is followed by a tobacco etch virus protease (TEV) sensitive sequence, that is responsible to separate the mass tag from the affinity tag, which was either the Strep-tag II sequence or biotin and were added for purification purposes. Chapter 1 presents a step-by-step protocol on how to design a mass tag family allowing for multiplexed analysis by LC-MS/MS. The multiplexing is achieved by developing an isobar mass tag family with four family members, which are chromatographically indistinguishable, but due to the mass encoding principles they fragment in distinct y-type ions with a mass difference of 1 or 2 Da each in MS2. Furthermore, it is explained how to covalently attach the mass reporter peptides onto ECM by the activated calcium-catalyzed blood coagulation transglutaminase factor XIII (FXIIIa). The lysine of mass reporter’s TG sequence (D-domain of insulin-like growth factor-I (IGF-I)) and a glutamine in fibronectin are covalently crosslinked by FXIIIa and build an isopeptide bond. Elevated levels of MMP release the mass reporters from ECM by recognizing the inter-positioned MSS. The designed mass reporters were able to monitor enzyme activity in an in vitro setting with cell-derived ECM, which was shown in Chapter 2. The modular structured mass reporters were investigated in a proof of concept study. First, the different modules were characterized in terms of their MMP responsiveness and their sensitivity to TEV protease and UV light. Then the FXIIIa-mediated coupling reaction was detailed and the successful coupling on ECM was visualized by an immunosorbent assay or confocal laser scanning microscopy. Finally, the immobilized mass reporters on ECM were incubated with MMP-9 to investigate their multiplexing ability of MMP activity. The cleaved mass reporter fragments were purified in three steps and mass tags were analyzed as mix of all four in LC-MS/MS. Chapter 3 describes the change from an immobilizing system as seen in chapter 1 and 2 to a soluble enzyme activity monitoring system that was applied in an osteoarthritic mouse model. Instead of the immobilizing TG sequence the C-terminal MMS was extended with two amino acids where one holds an azide moiety to perform a strain-promoted azide-alkyne cycloaddition to a high molecular weight dibenzocyclooctyne-polyethylene glycol (DBCO-PEG), which was chosen to retain the mass reporters at the injection site. Furthermore, the N-terminal affinity tag was extended with a 2.5 kDa PEG chain to increase the half-life of the mass reporter peptides after MMP release. The systems biocompatibility was proved but its enzyme monitoring ability in an in vivo setting could not be analyzed as samples degraded during shipping resulting from the Chinese customs blocking transport to Germany. In summary the diagnostic peptide system was developed in two variants. The immobilized version one from chapter 1 and 2 was designed to be covalently attached to ECM by the transglutaminase-mediated cross-linking reaction. In an in vitro setting the functionality of the mass reporter system for the detection of MMP activity was successfully verified. The second variant comprises of a soluble mass reporter system that was tested in an OA mouse model and showed biocompatibility. With these two designed systems this thesis provides a flexible platform based on multiplexed analysis with mass-encoded peptides to characterize cell culture models regarding their MMP activity, to deploy cell-derived ECM as endogenous depot scaffold and to develop a mass tag family that enables simultaneous detection of at least four mass tags.

Antimikrobielle Resistenzen stellen eine weltweite Herausforderung dar und sind mit einer hohen Morbidität und Mortalität verbunden. Die Letalitätsrate durch multiresistente Keime steigt stetig an, weshalb die WHO im Jahr 2017 eine Prioritätenliste resistenter Keime erstellte, die die Entwicklung neuer Antibiotika vorantreiben soll. Diese umfasst vornehmlich gramnegative Bakterien, da diese aufgrund ihres Zellaufbaus sowie diverser Resistenzmechanismen besonders widerstandsfähig gegenüber dem Angriff vieler Antibiotika sind. Einige grampositive Keime (z.B. S. aureus) stehen ebenfalls auf dieser Liste und stellen eine große Herausforderung für die Medizin dar. Infolgedessen ist die Entwicklung neuer Antiinfektiva mit neuen Angriffspunkten gegen resistente Pathogene zwingend nötig, um mit bisherigen Resistenzen umgehen zu können. Die vorliegende Arbeit beschäftigt sich mit der Entwicklung und Synthese von kovalent (reversibel) bindenden Inhibitoren der Enoyl-ACP-Reduktase FabI (Staphylococcus aureus, Escherichia coli) und der Thiolase FadA5 (Mycobacterium tuberculosis). Beide Enzyme sind essenziell für das Überleben des jeweiligen Bakteriums. FabI ist ein wichtiges und geschwindigkeitsbestimmendes Schlüsselenzym der Fettsäuresynthese Typ II diverser Bakterien. Hierbei werden wichtige Phospholipide hergestellt, die für den Aufbau der Zellmembran nötig sind. Schiebel et al. ist es gelungen, einen potenten Inhibitor für den Erreger S. aureus sowie E. coli zu entwickeln und zu charakterisieren. Ausgehend von dieser Verbindung wurde eine Substanzbibliothek mit verschiedenen „warheads“ hergestellt. Hierbei wurde die Verknüpfung zwischen dem Pyridon-Grundgerüst und der elektrophilen Gruppe sowie die über den Ether verknüpften aromatischen Ringsysteme variiert. Diese Verbindungen wurden hinsichtlich ihrer inhibitorischen Aktivität am jeweiligen Enzym getestet. Anschließend wurde von Verbindung 32 und 33, die jeweils eine gute Inhibition des Enzyms aufweisen, der IC50-Wert gemessen. Beide Verbindungen weisen eine 50-prozentige Reduktion der Enzymaktivität im mittleren nanomolaren Bereich auf. Zusätzlich wurde Verbindung 32 in einem sogenannten „jump-dilution“-Assay auf kovalente Inhibition getestet. Durch dieses Experiment konnte eine kovalente Inhibition des Enzyms ausgeschlossen werden. Die Reaktivität der eingesetzten „warheads“ wurde gegenüber einem Tripeptid mittels eines LC/MS-Iontrap-Systems bestimmt. Die untersuchten Verbindungen zeigten keine signifikante Reaktion mit der im Tripeptid eingebauten nukleophilen Aminosäure Tyrosin, deren Nukleophilie bei dem pH-Wert des Tests (pH = 8.2 und 10.8) nicht hoch genug ist. Um einen Einblick in den Bindemodus der Verbindungen zu erhalten, wurden ferner Kristallisationsversuche durchgeführt. Die erhaltenen Kristallstrukturen zeigen, dass die Verbindungen mit dem gewünschten Bindemodus am Zielenzym binden, aber eine kovalente Modifizierung des Tyrosins146 durch die eingesetzten „warheads“ aufgrund der großen Entfernung (6 Å zwischen elektrophiler Gruppe und Tyrosin146), unwahrscheinlich ist. Zusätzlich wurden die physikochemischen Eigenschaften (Stabilität, Wasserlöslichkeit und logP) der Verbindung 32 sowie Verbindung 33 charakterisiert. M. tuberculosis ist der Erreger der global verbreiteten Infektionskrankheit Tuberkulose (TB), die zu den zehn häufigsten Todesursachen weltweit gehört. Das Bakterium kann das im menschlichen Körper vorkommende Cholesterol metabolisieren und nutzt dessen Abbauprodukte als wichtige Kohlenstoffquelle. Die Thiolase FadA5 ist bei diesem Abbau ein wichtiges Enzym und konnte als potenzielles innovatives Target für neue Antibiotika definiert werden. Durch Dockingstudien konnten zwei potenzielle Leitstrukturen als Inhibitoren der Thiolase FadA5 identifiziert werden. Im Rahmen dieser Arbeit wurden die vorgeschlagenen Strukturen mit dem gewünschten „warhead“ synthetisiert und hinsichtlich ihrer inhibitorischen Aktivität gegenüber dem Enzym untersucht. Die Zielverbindungen zeigen keine signifikante Hemmung sowie kovalente Bindung über die eingesetzten „warheads“ an die Thiolase FadA5.

Microbial, mammalian, and plant cells produce and contain secondary metabolites, which typically are soluble in water to prevent cell damage by crystallization. The formation of ion pairs, for example, with carboxylic acids or mineral acids, is a natural blueprint to maintain basic metabolites in solution. Here, we aim at showing whether the mostly large carboxylates form soluble protic ionic liquids (PILs) with the basic natural product papaverine resulting in enhanced aqueous solubility. The obtained PILs were characterized by H-1-N-15 HMBC nuclear magnetic resonance (NMR) and in the solid state using X-ray powder diffraction, differential scanning calorimetry, and dissolution measurements. Furthermore, their supramolecular pattern in aqueous solution was studied by means of potentiometric and photometrical solubility, NMR aggregation assay, dynamic light scattering, zeta potential, and viscosity measurements. Thereby, we identified the naturally occurring carboxylic acids, citric acid, malic acid, and tartaric acid, as being appropriate counterions for papaverine and which will facilitate the formation of PILs with their beneficial characteristics, like the improved dissolution rate and enhanced apparent solubility.

The muscarinic M\(_1\) acetylcholine receptor is an important drug target for the treatment of various neurological disorders. Designing M\(_1\) receptor-selective drugs has proven challenging, mainly due to the high conservation of the acetylcholine binding site among muscarinic receptor subtypes. Therefore, less conserved and topographically distinct allosteric binding sites have been explored to increase M\(_1\) receptor selectivity. In this line, bitopic ligands, which target orthosteric and allosteric binding sites simultaneously, may provide a promising strategy. Here, we explore the allosteric, M1-selective BQCAd scaffold derived from BQCA as a starting point for the design, synthesis, and pharmacological evaluation of a series of novel bitopic ligands in which the orthosteric moieties and linker lengths are systematically varied. Since β-arrestin recruitment seems to be favorable to therapeutic implication, all the compounds were investigated by G protein and β-arrestin assays. Some bitopic ligands are partial to full agonists for G protein activation, some activate β-arrestin recruitment, and the degree of β-arrestin recruitment varies according to the respective modification. The allosteric BQCAd scaffold controls the positioning of the orthosteric ammonium group of all ligands, suggesting that this interaction is essential for stimulating G protein activation. However, β-arrestin recruitment is not affected. The novel set of bitopic ligands may constitute a toolbox to study the requirements of β-arrestin recruitment during ligand design for therapeutic usage.