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Copper perchlorophthalocyanine (CuPcCl16, CuC32N8Cl16, Pigment Green 7) is one of the commercially most important green pigments. The compound is a nanocrystalline fully insoluble powder. Its crystal structure was first addressed by electron diffraction in 1972 [Uyeda et al. (1972). J. Appl. Phys. 43, 5181–5189]. Despite the commercial importance of the compound, the crystal structure remained undetermined until now. Using a special vacuum sublimation technique, micron-sized crystals could be obtained. Three-dimensional electron diffraction (3D ED) data were collected in two ways: (i) in static geometry using a combined stage-tilt/beam-tilt collection scheme and (ii) in continuous rotation mode. Both types of data allowed the crystal structure to be solved by direct methods. The structure was refined kinematically with anisotropic displacement parameters for all atoms. Due to the pronounced crystal mosaicity, a dynamic refinement was not feasible. The unit-cell parameters were verified by Rietveld refinement from powder X-ray diffraction data. The crystal structure was validated by many-body dispersion density functional theory (DFT) calculations. CuPcCl16 crystallizes in the space group C2/m (Z = 2), with the molecules arranged in layers. The structure agrees with that proposed in 1972.
Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.
The DNA damage response (DDR) is a vast network of molecules that preserves genome integrity and allow the faithful transmission of genetic information in human cells. While the usual response to the detection of DNA lesions in cells involves the control of cell-cycle checkpoints, repair proteins or apoptosis, alterations of the repair processes can lead to cellular dysfunction, diseases, or cancer. Besides, cancer patients with DDR alterations often show poor survival and chemoresistance. Despite the progress made in recent years in identifying genes and proteins involved in DDR and their roles in cellular physiology and pathology, the question of the involvement of DDR in metabolism remains unclear. It remains to study the metabolites associated with specific repair pathways or alterations and to investigate whether differences exist depending on cellular origin. The identification of DDR-related metabolic pathways and of the pathways that cause metabolic reprogramming in DDR-deficient cells may produce new targets for the development of new therapies.
In this thesis, nuclear magnetic resonance spectroscopy (NMR) was used to assess the metabolic consequence of the loss of two central DNA repair proteins with importance in diseases context, ATM and RNase H2, in haematological cells. An increase in intracellular taurine was found in RNase H2- and ATM-deficient cells compared to wild-type cells for these genes and in cells after exposition to a source of DNA damage. The rise in taurine does not appear to result from an increase in its biosynthesis from cysteine, but more likely from other cellular processes such as degradation pathways.
Overall, evidence for metabolic reprogramming in haematological cells with faults in DNA repair resulting from ATM or RNase H2 deficiencies or upon exposition to a source of DNA damage is presented in this study.
Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
(2021)
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.
Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
(2021)
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases.
Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions.
In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo.
In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14.
Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation.
...
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
Sphingosin 1 Phosphat (S1P) ist ein wichtiger Lipidmediator, der über G Protein gekoppelte Rezeptoren und intrazelluläre Wirkungen vielfältige Wirkungen auslöst und eine Rolle bei der Lymphozytenzirkulation, der Erhaltung der endothelialen Barriere, bei Entzündungsprozessen und Tumorwachstum spielt. Die S1P Lyase (Sgpl1) katalysiert den irreversiblen Abbau von S1P und damit den letzten Schritt des Sphingolipidkatabolismus‘. Ein Fehlen der Sgpl1 bewirkt eine Akkumulation von S1P und anderen Sphingolipiden im Blut und Gewebe, was multiple Organschäden zur Folge hat. Menschen mit S1P Lyase Insuffizienz Syndrom (SPLIS) leiden insbesondere unter steroidresistentem nephrotischem Syndrom, Nebennierenrinden-insuffizienz und neurologischen Störungen. Weitere mögliche Symptome sind Lymphopenie, Hautveränderungen und Dyslipidämien. S1P Lyase defiziente Mäuse weisen sehr ähnliche Organschädigungen auf.
An Sgpl1 Knockoutmäusen war zuerst die massive Akkumulation nicht nur von Sphingolipiden, sondern auch von Cholesterin und Triglyceriden in Blut und Leber aufgefallen. Auch bei SPLIS Patienten wurde eine Hypercholesterinämie beobachtet. Um die Kreuzregulation des Sphingolipid- und Cholesterinmetabolismus besser zu verstehen, sollte die Rolle der Sgpl1 in der Leber, dem Hauptort des Lipidmetabolismus, untersucht werden. Hierzu sollte ein Mausmodell mit einem hepatozytenspezifischen Sgpl1 Knockout (Sgpl1HepKO) etabliert und charakterisiert werden. Dies wurde durch Kreuzen von Sgpl1fl/fl-Mäusen mit Mäusen, welche die Cre-Rekombinase unter dem Albuminpromoter exprimierten, erreicht. Die basale Charakterisierung zeigte, dass diese Mäuse im Gegensatz zu globalen Sgpl1 Knockoutmäusen sowohl im Alter von acht Wochen, als auch im Alter von acht Monaten einen unauffälligen Phänotyp aufwiesen. Das äußere Erscheinungsbild inklusive Leber und Körpergewicht, das Blutbild, die Leberenzyme sowie die Histologie der Leber waren unverändert. Die Analyse der Leberlipide mit Hilfe von Hochleistungsflüssigkeits-chromatographie gekoppelt mit einer Tandem Massenspektrometrie zeigte eine signifikante Akkumulation (≈1,5 2 fach) von S1P, Sphingosin und Ceramiden, aber nicht von Glucosylceramiden und Sphingomyelin in der Leber. Messungen im Plasma zeigten eine Erhöhung mehrerer Ceramide, während der S1P Spiegel normal war. Ferner zeigten Untersuchungen der Galle signifikant erhöhte Konzentrationen an S1P, Dihydro S1P und Glucosylceramiden, jedoch unveränderte Ceramide. Die Ergebnisse legen folgende Schlussfolgerungen nahe: 1. In der Leber kann mit Hilfe von Ceramidsynthasen akkumulierendes Sphingosin in Ceramide umgewandelt werden, welche anschließend ins Blut sezerniert und letztendlich vermutlich von anderen Zellen verstoffwechselt werden. Außerdem ist nicht ausgeschlossen, dass S1P ebenfalls ins Blut sezerniert und dort effektiv abgebaut wird, so dass die S1P Konzentration im Plasma unverändert bleibt. 2. S1P sowie Glucosylceramide werden an die Galle abgegeben und ausgeschieden. 3. Die Sgpl1 in der Leber ist nicht essentiell für die Regulation des Plasma S1Ps, was zuvor vermutet worden war
Eine Analyse der Sterole zeigte in Sgpl1HepKO Mäusen erhöhte Spiegel an Cholesterin und Desmosterol in der Leber. In Übereinstimmung mit der erhöhten Proteinexpression des low density lipoprotein (LDL ) Rezeptors und erniedrigten Konzentrationen des LDL Cholesterins im Plasma, deuten diese Daten auf eine erhöhte Aufnahme von LDL Cholesterin durch die Leber hin. Untersuchungen in der Leber sowie mit primären Hepatozyten zeigten im Gegensatz zu globalen Sgpl1 Knockoutmäusen keine Veränderungen der Peroxisomen-Proliferator-aktiviertem Rezeptor γ Expression. Weitere Gene mit zentraler Rolle wie der Liver X receptor oder die Fettsäuresynthase, waren ebenfalls nicht reguliert. Dieser im Vergleich zu globalen Sgpl1-Knockoutmäusen milde Phänotyp lässt sich durch die deutlich geringere Akkumulation von Sphingolipiden aufgrund der oben beschriebenen Kompensations-mechanismen in Sgpl1HepKO Mäusen erklären.
In weiteren Untersuchungen sollten die Auswirkungen einer Sgpl1-Defizienz an Fibroblasten untersucht werden. Hierzu standen embryonale Fibroblasten aus Sgpl1 Knockoutmäusen zur Verfügung (Sgpl1-/- MEFs). In einer Kooperation mit Dr. Janecke von der Universität Innsbruck standen außerdem humane Fibroblasten eines SPLIS Patienten zur Verfügung.
An Sgpl1-/- MEFs war zuvor eine gestörte Calciumhomöostase festgestellt worden, welche sich durch eine erhöhte zytosolische Calciumkonzentration und vermehrte Calciumspeicherung im Endoplasmatischen Retikulum und in Lysosomen auszeichnete. Die Plasmamembran-Calcium ATPase (PMCA) trägt an Fibroblasten entscheidend zur Regulation der zytosolischen Calciumkonzentration bei. Ihre Expression auf Proteinebene war jedoch in Sgpl1-/- MEFs nicht verändert. Im Rahmen dieser Arbeit wurde durch eine Immunfärbung erstmals festgestellt, dass die PMCA in Sgpl1-/- MEFs nicht vollständig an der Plasmamembran lokalisiert war. Dies könnte der Grund für die erhöhte zytosolische Calciumkonzentration in den Zellen sein. ...
Additive manufacturing or 3D printing as an umbrella term for various materials processing methods has distinct advantages over many other processing methods, including the ability to generate highly complex shapes and designs. However, the performance of any produced part not only depends on the material used and its shape, but is also critically dependent on its surface properties. Important features, such as wetting or fouling, critically depend mainly on the immediate surface energy. To gain control over the surface chemistry post-processing modifications are generally necessary, since it′s not a feature of additive manufacturing. Here, we report on the use of initiator and catalyst-free photografting and photopolymerization for the hydrophilic modification of microfiber scaffolds obtained from hydrophobic medical-grade poly(ε-caprolactone) via melt-electrowriting. Contact angle measurements and Raman spectroscopy confirms the formation of a more hydrophilic coating of poly(2-hydroxyethyl methacrylate). Apart from surface modification, we also observe bulk polymerization, which is expected for this method, and currently limits the controllability of this procedure.
The stress-dependent dynamics of Saccharomyces cerevisiae tRNA and rRNA modification profiles
(2021)
RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.
The prevalence and specificity of local protein synthesis during neuronal synaptic plasticity
(2021)
To supply proteins to their vast volume, neurons localize mRNAs and ribosomes in dendrites and axons. While local protein synthesis is required for synaptic plasticity, the abundance and distribution of ribosomes and nascent proteins near synapses remain elusive. Here, we quantified the occurrence of local translation and visualized the range of synapses supplied by nascent proteins during basal and plastic conditions. We detected dendritic ribosomes and nascent proteins at single-molecule resolution using DNA-PAINT and metabolic labeling. Both ribosomes and nascent proteins positively correlated with synapse density. Ribosomes were detected at ~85% of synapses with ~2 translational sites per synapse; ~50% of the nascent protein was detected near synapses. The amount of locally synthesized protein detected at a synapse correlated with its spontaneous Ca2+ activity. A multifold increase in synaptic nascent protein was evident following both local and global plasticity at respective scales, albeit with substantial heterogeneity between neighboring synapses.
Bezüglich der Arzneimittelforschung galt für sehr lange Zeit das Paradigma "ein Gen, ein Medikament, eine Krankheit". In jüngerer Zeit ändert sich dieses Paradigma jedoch auf Grund von redundanten Funktionen und alternativen sich kompensierenden Signalmustern, die insbesondere bei Krebserkrankungen vorherrschend sind. Daher kann die logische Konsequenz nur sein, Multi-Target-Strategien gegenüber Single-Target-Ansätzen in Betracht zu ziehen. Auf Grund der Schwierigkeit, mit einer Kombination von zwei Einzelwirkstoffen, in diesem Fall BET- und HDAC-Inhibitoren eine konsistente Biodistribution und Pharmakokinetik zu erreichen, wurde nach Einzelmolekülen gesucht, die mehrere inhibitorische Aktivitäten aufweisen. Dies wurde hier zunächst durch die einfache Konjugation von zwei unterschiedlichen Pharmakophoren erreicht.
Insgesamt wurden vier verschiedene Liganden dieses Typs synthetisiert und einer von ihnen, Verbindung 14, zeigte sehr vielversprechende Ergebnisse. 14 vereint den BET Inhibitor JQ1- mit dem HDAC Inhibitor CI994 und hat eine hemmende Wirkung sowohl gegen BRD4- als auch HDAC-Proteine wie durch DSF- und nanoBRET-Assay gezeigt werden konnte. Außerdem zeigten in vitro Assays in PDAC-Zellen, dass 14 ein noch potenterer dualer BET/HDAC-Inhibitor ist als die Kombination aus JQ1 und CI994. Während die Effekte von 14 auf das BETi-Antwortgen MYC denen von JQ1 ziemlich ähnlich sind, sind insbesondere die HDAC-inhibitorischen Effekte nachhaltiger und verstärkt, wahrscheinlich aufgrund einer längeren Verweildauer von 14 auf HDAC als dies bei CI994 der Fall ist. Dies ist durch das hohe Niveau der acetylierten Lysine von Histon H3 im Western Blot erkennbar. Dieses veränderte Expressionsverhalten hatte einen großen Einfluss auf das Zellwachstum und überleben in allen getesteten PDAC-Zelllinien. Hier wurde die Überlegenheit von 14 gegenüber der gleichzeitigen Behandlung der Zellen mit JQ1 und CI994 sehr deutlich. Wurden PDAC-Zellen mit dem dualen Inhibitor 14 behandelt, hatte dies ein geringeres Wachstum und Überleben der Krebszellen zur Folge als mit beiden ursprünglichen Molekülen, unabhängig davon, ob diese einzeln oder simultan verabreicht wurden. Außerdem wurde 14 mit Gemcitabin, einem gut verträglichen Chemotherapeutikum, kombiniert, dass bei PDAC allein nur eine begrenzte Aktivität aufweist. Es stellte sich heraus, dass die Reihenfolge, in der die Medikamente verabreicht werden, einen großen Einfluss auf die Effektivität hatte. Der durch 14 induzierte Stopp des Zellzyklus verhindert den Einbau von Gemcitabin in die DNA, wenn 14 vor oder gleichzeitig mit Gemcitabin verabreicht wird. Wenn jedoch die Behandlung mit 14 nach der Verabreichung von Gemcitabin folgt, wird der durch Gemcitabin induzierte S-Phasen-Arrest und Replikationsstress aufrechterhalten. Im Vergleich zu den meisten früheren Studien, die sich mit dualen BET/HDAC-Inhibitoren beschäftigten, ist dies eine große Verbesserung, da es bisher keinen signifikanten Unterschied zwischen der Verwendung eines dualen BET/HDAC-Inhibitors und der Kombination von zwei Einzelinhibitoren gab.
Als Proof of Concept unterstützten die Daten weitere Bemühungen zur Entwicklung zusätzlicher dualer BET/HDAC-Inhibitoren. Daher wurden zwei weitere Generationen dualer BET/HDAC Inhibitoren entwickelt, die jedoch bisher nicht an die Eigenschaften von 14 anknüpfen konnten. Vor allem die 3. Generation bietet jedoch Raum für Optimierungen, so dass hier möglicherweise noch ein potenter dualer Inhibitor zu finden ist. Sollte es in Zukunft einen zugelassenen dualen BET/HDAC-Inhibitor geben, ist es jedoch nicht unwahrscheinlich, dass keine der hier verwendet BET inhibierenden Strukturen verwendet werden, aber Struktur des HDAC inhibierenden Teils immer noch vergleichbar ist. Der Grund dafür ist, dass die HDAC Inhibitoren größtenteils relativ einfach aufgebaut. So lange das wichtigste, die zinkbindende Gruppe vorhanden ist, scheint der Linker sowie die Capping-Gruppe zweitranging zu sein. Die größere Herausforderung wird vermutlich die Suche nach dem passenden BET Inhibitor sein und die Wahlmöglichkeiten sind schon jetzt vielfältig.
Generell lässt sich sagen, dass die Idee der dualen BET/HDAC-Inhibitoren äußerst vielversprechend und es wert ist, weiter verfolgt zu werden. Dies liegt vor allem an den guten Testergebnissen, die mit Verbindung 14 erzielt wurden. Mit Hilfe dieser Art von Inhibitoren könnte es in Zukunft möglich sein, die Überlebensrate von PDAC-Patienten zu erhöhen, wenn nicht als alleiniges Medikament, so vielleicht als Zusatz zur Chemotherapie. Darüber hinaus scheint der Einsatz von dualen BET/HDAC-Inhibitoren nicht nur auf die Behandlung von PDAC beschränkt zu sein und kann auch bei anderen Krebsarten angewendet werden. NMC zum Beispiel ist ein ebenso seltener wie tödlicher Subtyp des schlecht differenzierten Plattenepithelkarzinoms und zeichnet sich durch eine Fusion des NUT-Gens mit BRD4 aus, wodurch es potenziell anfällig für eine BET-Inhibition ist. Tatsächlich zeigte 14 auch hier einen größeren positiven Effekt auf die getesteten NMC-Zellen als JQ1 oder CI994 und veranlasste die Zellen unter anderem zur Differenzierung. ...
Standard biorelevant media reflect the average gastrointestinal (GI) physiology in healthy volunteers. The use of biorelevant media in in vitro experiments has become an important strategy to predict drug behaviour in vivo and is often combined with in silico tools in order to simulate drug plasma profiles over time. In addition to the healthy population, the effects of disease state or co-administration of other drugs on plasma profiles must be considered to assure drug efficacy and safety. Thus, there is a need for a more accurate representation of the human GI physiology when it is altered by disease or co-administered drugs in in vitro dissolution experiments.
This thesis focused on the development of biorelevant media and dissolution tests reflecting GI physiology in circumstances where the gastric pH is elevated. Diseases linked to an elevated gastric pH are hypochlorhydria and achlorhydria, but these days treatment with acid-reducing agents (ARAs) is the single greatest cause of elevation in gastric pH. pH-dependent drug-drug interactions (DDIs) with ARAs are frequent, as the ARAs are used in a number of diseases using a variety of drugs. As the drugs currently on the market are often poorly soluble and ionisable, their dissolution is highly dependent on the pH of the GI tract, especially the gastric pH.
The thesis research consisted of several steps. In the first step, physiological changes in the human GI tract during the therapy with ARAs were identified. Parameters of the standard biorelevant gastric medium FaSSGF were adjusted to the identified changes to reflect the impact of ARA co-administration on the gastric physiology. The media aim to assess the potential extent of the ARA impact on gastric physiology by introducing biorelevant media pairs, ARA pH 4 and pH 6 media, of which one reflects a lesser, and the other a stronger impact of ARAs.
In the second step these ARA media were implemented in in vitro dissolution set-ups.
The dissolution of poorly soluble ionisable drugs was assessed using one-stage, two-stage and transfer model set-ups, as well as using a more evolved in vitro system TIM-1. Comparison of results from dissolution set-ups using the standard, low pH, gastric biorelevant medium FaSSGF (pH 1.6 or 2), and the same set-ups using ARA pH 4 and pH 6 media, shows a decrease in dissolution rate and extent for weakly basic compounds PSWB 001 and dipyridamole, and an increase in rate and extent of dissolution for the weakly acidic compound raltegravir potassium, when the gastric pH is elevated. Due to different physicochemical properties, the extent of the impact of physiological changes during ARA therapy (when either ARA pH 4 or pH 6 medium is selected) on dissolution varied among the model drugs. Thus, the bracketing approach, which considers a range of the possible ARA co-administration impact on drug dissolution, was confirmed to be best practice in assessing the impact of ARAs.
In the third step, dissolution data from in vitro experiments with ARA media was implemented into in silico models. The predictions using various in silico model approaches in Simcyp™ Simulator (minimal and full PBPK model, dissolution input using DRM and DLM) successfully bracketed in vivo data on drug administration during ARA therapy and correctly predicted an overall decrease in plasma concentration for the two model weakly basic compounds and an increase in plasma concertation for the model weakly acidic compound.
In all assessed scenarios, the ARA methods proved to be an essential part of evaluating and predicting the impact of ARAs on drug pharmacokinetics, and appropriately predicted the extent of a possible impact of ARAs on the drug plasma profiles. Thus, the ARA biorelevant media and dissolution tests were demonstrated to be valuable tools reflecting administration of drugs when the gastric pH is elevated and able to predict the impact of ARA therapy on drug administration.
The ability to evaluate the impact of human (patho) physioloy on drug behaviour in the gastrointestinal tract is of great importance, as the GI conditions play a significant role in drug release and absorption. Thus, there is great interest on the part of the pharmaceutical industry and regulatory agencies to develop best practices in this field, especially for pH-dependent DDIs. The media and dissolution tests developed in this thesis are biorelevant methods appropriate for evaluation of the impact of elevated gastric pH on drug efficacy and safety. Such methods, used as a risk assessment tool, in connection with evaluation of the efficacy window and potential toxicity, may help to increase confidence about decisions as to whether a pH-effect will occur and whether it is relevant or not, prior to conducting clinical studies. They may also enable changes in inclusion/exclusion criteria during recruiting for large-scale efficacy trials. In fact, the biopharmaceutic approach to drug development is becoming standard practice on a number of fronts, including metabolic DDIs, renal and hepatic insufficiency, powering decision-making process and possibly even waiving certain types of clinical studies.
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Bacteria are true artists of survival, which rapidly adapt to environmental changes like pH shifts, temperature changes and different salinities. Upon osmotic shock, bacteria are able to counteract the loss of water by the uptake of potassium ions. In many bacteria, this is accomplished by the major K+ uptake system KtrAB. The system consists of the K+-translocating channel subunit KtrB, which forms a dimer in the membrane, and the cytoplasmic regulatory RCK subunit KtrA, which binds non-covalently to KtrB as an octameric ring. This unique architecture differs strongly from other RCK-gated K+ channels like MthK or GsuK, in which covalently tethered cytoplasmic RCK domains regulate a single tetrameric pore. As a consequence, an adapted gating mechanism is required: The activation of KtrAB depends on the binding of ATP and Mg2+ to KtrA, while ADP binding at the same site results in inactivation, mediated by conformational rearrangements. However, it is still poorly understood how the nucleotides are exchanged and how the resulting conformational changes in KtrA control gating in KtrB is still poorly understood.
Here,I present a 2.5-Å cryo-EM structure of ADP-bound, inactive KtrAB, which for the first time resolves the N termini of both KtrBs. They are located at the interface of KtrA and KtrB, forming a strong interaction network with both subunits. In combination with functional and EPR data we show that the N termini, surrounded by a lipidic environment, play a crucial role in the activation of the KtrAB system. We are proposing an allosteric network, in which an interaction of the N termini with the membrane facilitates MgATP-triggered conformational changes, leading to the active, conductive state.
The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.
T-cell development is a highly dynamic and stepwise process comprimising T lineage commitment, T-cell receptor (TCR) gene rearrangements and subsequent selection. From a quantitative point of view, only a few hundred progenitor cells migrate from the bone marrow into the thymus. Developing thymocytes (termed double negative (DN), CD4-CD8-) can be further divided into DN1-4 cells based on the expression of CD25 and CD44. These developmental events are interspersed by proliferative bursts which ultimately lead to the generation of millions of double positive (DP, CD4+CD8+) thymocytes that then undergo selection. As a consequence, a proportion of naïve T-cells evolves to ensure adaptive, but not autoreactive immunity.
Previous studies of our lab focused on the quantification of thymus colonization and identified thymus entry to be dependent on expression of the chemokine receptors CCR7 and CCR9 (Krueger et al., 2010; Ziętara et al., 2015). CCR7/9 double knockout (DKO) mice are almost completely devoid of the most immature thymocyte populations (DN1 and DN2), but show near normal DN3 cellularity. Interestingly, a similar defect during early development but a virtually complete recovery of later stages and total thymocyte numbers was also observed in thymi of miR-17~92 deficient mice. Here, a failure of prethymic IL-7 signaling dampens early T-cell development (Regelin et al., 2015). For this reason, we hypothesized a tight regulation of thymocyte population size through alterations in the underlying cell cycle kinetics.
In this thesis, we employed in vivo single- and dual-nucleoside pulse labeling combined with determination of DNA replication over time in different WT thymocyte subsets at steady-state. Based on this, we assessed alterations in cell cycle kinetics of CCR7/9 and miR-17~92 defcicient mice and identified compensatory mechanisms of thymocytes on the level of cell cycle phase distribution and cell cycle speed. In addition, single-cell RNA sequencing helped to obtain information on cell cycle dynamics of early thymocyte subsets, exemplarily shown for WT and CCR7/9 DKO mice. Lastly, we performed cell cycle analyses in a model of endogenous thymic repair upon sublethal total body irradiation which provided insight into intrathymic cell cycle regulation as an adjustable system to re-establish normal thymus cellularity.
In the second part of the thesis, we addressed the role of miR-21 in the thymus. In various studies, we and others identified miRNAs as key posttranscriptional regulators of the immune system and especially for T-cell development (Regelin et al. 2015; Mildner et al. 2017; Li et al. 2007; Ebert et al. 2009; Ziętara et al. 2013; Schaffert et al. 2015). The dynamic expression of miR-21 during T-cell development (Neilson et al. 2007; Kirigin et al. 2012; Kuchen et al. 2010) prompted us to hypothesize that miR-21 has a regulatory function in the thymus. A miR 21-knockout mouse model allowed us to study the role of this miRNA for the development of T-cells in the thymus and the maintenance of T-cells in the periphery. In addition, we performed competitive bone marrow chimera experiments in the context of miR-21 deficiency and overexpression. Further insights were provided by exploring the function of miR-21 in negative selection in vivo as well as in T-cell differentiation in coculture experiments in vitro. To unravel implications of miR-21 to regulate cellular stress responses, we assessed the contribution of miR-21 in a model of endogenous regeneration of the thymus after sublethal irradiation. We could not provide evidence for a prominent role for miR-21 during T-cell development. Together, our experiments revealed that miR-21 is largely dispensable for physiologic T-cell development despite high and dynamic expression in the thymus (Kunze Schumacher et al., 2018). The apparent discrepancy between dynamic expression but lack of a regulatory function in the thymus led us to conclude that miR-21 is rather fine tuning T-cell responses than controlling a developmental event.
The specific and precise arrangement of proteins and biomolecules in 3D is an important prerequisite for the study of cell migration, cellular signal transduction and the production of artificial tissue. In a variety of research approaches, proteins have been immobilized on rigid surfaces such as glass or gold to observe protein-protein or protein-cell interactions. While these commonly used analytical platforms offer advantages such as rapid washing steps and easy use, due to their rigidity and two-dimensionality, they cannot replicate the extracellular matrix (ECM) the native environment of cells. This severe deviation from the natural environment results in significant changes in cell structure and cellular processes such as the polarization of the cell, its morphology, and signal transduction. In order to maintain the functionality of the immobilized proteins, it is also enormously important that the proteins are oriented and anchored in the material under mild conditions.
An immobilization strategy that makes this possible is bioaffinity. For this, the specific interaction of a biomolecule with an interaction partner anchored on a surface is used to immobilize the biomolecule. Such an interaction is for example the nitrilotriacetic acid (NTA)/His-tag binding. NTA is a chelator molecule that, when bound to divalent metal ions such as Ni(II), forms an octahedral complex with oligohistidines. The oligo histidine-tag can be competed out of the complex by free histidine or imidazole due to structural similarity. This is exploited in immobilized metal affinity chromatography (IMAC). The binding of a monoNTA/His-tag complex (KD=10 µM) is not stable enough to be used for immobilizations. Therefore, multivalent variants of the chelator were developed, like trisNTA which has a high affinity for His6 tagged proteins (KD= 10 nM). The PA-trisNTA developed in a preliminary work was the first light-activatable system based on the trisNTA chelator head.
The aim of this work was to synthesize a new two-photon (2P) activatable trisNTA (TPA trisNTA) interaction molecule, to analyze its photophysical characteristics and to apply it for two- and three dimensional (2D/3D) biomolecule patterning. The final goal was to use TPA trisNTA for cellular applications in order to manipulate membrane protein organization. Therefore, TPA trisNTA was designed to maintain a stable autoinhibition enabling the immobilization of proteins under physiological conditions with high precision in the x/y, as well as z dimension only upon light activation. 2P activation brings some outstanding advantages: i) the use of near-infrared (NIR) light is less harmful to cells compared to ultraviolet (UV) light, ii) the longer wavelength allows the radiation to penetrate deeper into tissues, iii) the precision of focal irradiation is more accurate because only a focal volume (about 1 fL) is excited and, unlike UV light, scattered light does not lead to activation.
Several backbones for TPA-trisNTA were considered as 2P cleavable groups due to their 2P absorption ability and small size: 3 nitrodibenzofuran (NDBF), 6 bromo 7 hydroxycoumarin (Bhc), and 7 diethylaminocoumarin (DEAC). Initially, suitable synthetic routes were developed for the respective carbaldehydes, since these represented an important intermediate for both the construction of amino acid (aa) derivatives as well as ß hydroxy acids. ß Hydroxy acids were important intermediates because their photocleavage differs from aa derivatives. To establish the conversion from carbaldehydes to hydroxy acids via Reformatsky reaction, commercially available carbaldehydes of the nitroveratral (NV) or nitropiperonal (NP) group were used in addition. The conversion of NDBF, NV, NP proved to be difficult, whereas the ß-hydroxy acid was successfully synthesized from Bhc as well as from DEAC.
Starting from DEAC ß hydroxy acid, a Fmoc protected amino acid derivative was synthesized. To ensure high cleavage efficiency, the DEAC ß hydroxy acid was linked to monoFmoc ethylenediamine through a carbamate linker. Subsequently, the photocleavable group was successfully incorporated into the linker of TPA-trisNTA by solid-phase peptide synthesis (SPPS).
The functional principle of TPA-trisNTA, similar to PA-trisNTA, is based on the autoinhibition of the multivalent chelator head trisNTA, which is linked to an intramolecular oligohistidine sequence by a peptide linker. In presence of Ni(II) ions, trisNTA forms a metal ion-mediated complex with histidine, causing TPA-trisNTA to self-inactivate. The cleavage site is the DEAC based photocleavable amino acid. In contrast to PA-trisNTA, the incorporation of two photocleavable amino acids was omitted. Instead, only one photocleavable DEAC was incorporated in front of the His tag. To avoid a second DEAC group within the His tag, a His5 tag was used instead of an His6 tag. It is known from preliminary work that a His5 tag is sufficient to maintain autoinhibition in the presence of His6-tagged proteins of interest (POIs), but can be displaced from the complex after light-driven cleavage of the peptide backbone. Placement of a cysteine in the peptide linker between the trisNTA and the DEAC group allowed for permanent surface anchoring after photocleavage of the linker.
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Treatment of hexachloropropene (Cl2C[double bond, length as m-dash]C(Cl)–CCl3) with Si2Cl6 and [nBu4N]Cl (1 : 4 : 1) in CH2Cl2 results in a quantitative conversion to the trisilylated, dichlorinated allyl anion salt [nBu4N][Cl2C[double bond, length as m-dash]C(SiCl3)–C(SiCl3)2] ([nBu4N][1]). Tetrachloroallene Cl2C[double bond, length as m-dash]C[double bond, length as m-dash]CCl2 was identified as the first intermediate of the reaction cascade. In the solid state, [1]− adopts approximate Cs symmetry with a dihedral angle between the planes running through the olefinic and carbanionic fragments of [1]− of C[double bond, length as m-dash]C–Si//Si–C–Si = 78.3(1)°. One-electron oxidation of [nBu4N][1] with SbCl5 furnishes the distillable blue radical 1˙. The neutral propene Cl2C[double bond, length as m-dash]C(SiCl3)–C(SiCl3)2H (2) was obtained by (i) protonation of [1]− with HOSO2CF3 (HOTf) or (ii) H-atom transfer to 1˙ from 1,4-cyclohexadiene. Quantitative transformation of all three SiCl3 substituents in 2 to Si(OMe)3 (2OMe) or SiMe3 (2Me) substituents was achieved by using MeOH/NMe2Et or MeMgBr in CH2Cl2 or THF, respectively. Upon addition of 2 equiv. of tBuLi, 2Me underwent deprotonation with subsequent LiCl elimination, 1,2-SiMe3 migration and Cl/Li exchange to afford the allenyl lithium compound Me3Si(Li)C[double bond, length as m-dash]C[double bond, length as m-dash]C(SiMe3)2 (Li[4]), which is an efficient building block for the introduction of Me, SiMe3, or SnMe3 (5) groups. The trisilylated, monochlorinated allene Cl3Si(Cl)C[double bond, length as m-dash]C[double bond, length as m-dash]C(SiCl3)2 (6), was obtained from [nBu4N][1] through Cl−-ion abstraction with AlCl3 and rearrangement in CH2Cl2 (1˙ forms as a minor side product, likely because the system AlCl3/CH2Cl2 can also act as a one-electron oxidant).