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In den letzten 20 Jahren haben sich zunehmend Schülerlabore an Universitäten und Forschungszentren etabliert, um naturwissenschaftliche Kompetenzen von Schülern/innen aufzubauen und zu fördern. Das wachsende Feld der Schülerlaborforschung zeigt allerdings auf, dass die Eingangsvoraussetzungen, mit denen die Schüler/innen an der naturwissenschaftlichen Lernumgebung teilnehmen, einen starken Einfluss auf die Annahme sowie die Entwicklung von interessens-, selbstkonzept- und motivationsbezogenen Persönlichkeitsmerkmalen haben können. Diese Erkenntnisse lenken den Blick auf die Entwicklungsumwelt der Familien, in der die Eltern von Geburt an auf die kindliche Persönlichkeitsentwicklung und Lernprozesse einwirken. Die Integration des vielversprechenden familiären Kontexts in eine naturwissenschaftliche Lernumgebung wird seit 2008 anhand des Eltern-Kind-Projekts KEMIE® an der Ruhr-Universität Bochum umgesetzt, indem Eltern-Kind-Paare gemeinsam an alltagsnahen naturwissenschaftlichen Phänomenen experimentieren. Seit 2016 wird KEMIE® zudem an der Goethe-Universität Frankfurt am Main im Goethe-Schülerlabor Chemie durchgeführt.
Am Beispiel des Frankfurter KEMIE®-Projekts will die vorliegende Arbeit das Potential von Eltern-Kind-Interaktionen für naturwissenschaftliche Lernprozesse untersuchen. Mithilfe der Grounded-Theory-Methodologie wurde das zentrale Forschungsdesiderat der Eltern-Kind-Interaktion über die Projektjahre 2016/17 und 2017/18 herausgestellt sowie mögliche Erhebungsmethoden pilotiert. Im Projektjahr 2018/19 konnte dieses schließlich in einer Mixed-Methods-Studie multiperspektivisch beforscht werden. Dafür wurden Interviews, Beobachtungen und Pre-Posttest-Daten von 46 teilnehmenden Eltern-Kind-Paaren sowie weitere Kontrollgruppendaten von 202 Frankfurter Familien erhoben.
Um zunächst die naturwissenschaftliche Lernumgebung zu beschreiben, wurden die vorherrschenden Einflussfaktoren identifiziert, die zugehörigen Phänomene beschrieben sowie die prozess- und personenbezogenen Verhaltensmuster herausgestellt. Über letztere können die Eltern-Kind-Interaktionen in das SELE-Modell elterlicher Unterstützungsmaßnahmen eingeordnet werden, wobei sich das Ermöglichen von Selbsttätigkeit als herausragendes Merkmal für die naturwissenschaftliche Lernumgebung manifestiert. Zudem zeigt sich, dass das Projekt positive Effekte auf naturwissenschaftsbezogene Werteorientierungen der Kinder sowie deren Interesse an den Naturwissenschaften hat.
Anhand der Entwicklung eines induktiven Beobachtungsschemas, konnten die Familien schließlich über die Verhaltensmerkmale der Eltern-Kind-Interaktion gruppiert und Zusammenhänge zu sowohl den Vorbedingungen als auch den Konsequenzen für die psychischen Dispositionen der Kinder aufgedeckt werden. Erste Erkenntnisse dieser explorativen Erhebung zeigen, dass das akademische Selbstkonzept und die naturwissenschaftsbezogenen Werteorientierungen der Eltern und Kinder die Verhaltensmuster in der naturwissenschaftlichen Umgebung determinieren. Auf der anderen Seite können die Kinder gerade hinsichtlich dieser beiden Faktoren am meisten profitieren. Dabei können diese positiven Effekte auf eine Regulation seitens der Eltern, das gemeinsame Ausführen von Tätigkeiten sowie den Einbezug in die Gestaltung der Lernumgebung zurückgeführt werden.
We performed an X-ray crystallographic study of complexes of protein kinase PIM-1 with three inhibitors comprising an adenosine mimetic moiety, a linker, and a peptide-mimetic (d-Arg)6 fragment. Guided by the structural models, simplified chemical structures with a reduced number of polar groups and chiral centers were designed. The developed inhibitors retained low-nanomolar potency and possessed remarkable selectivity toward the PIM kinases. The new inhibitors were derivatized with biotin or fluorescent dye Cy5 and then applied for the detection of PIM kinases in biochemical solutions and in complex biological samples. The sandwich assay utilizing a PIM-2-selective detection antibody featured a low limit of quantification (44 pg of active recombinant PIM-2). Fluorescent probes were efficiently taken up by U2OS cells and showed a high extent of co-localization with PIM-1 fused with a fluorescent protein. Overall, the developed inhibitors and derivatives represent versatile chemical tools for studying PIM function in cellular systems in normal and disease physiology.
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.
Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood—restoring the blood's natural coagulation capability.
Medicinal plants represent a big reservoir for discovering new drugs against all kinds of diseases including inflammation. In spite the large number of promising anti-inflammatory plant extracts and isolated components, research on medicinal plants proves to be very difficult. Based on that background this review aims to provide a summarized insight into the hitherto known pharmacologically active concentrations, bioavailability, and clinical efficacy of boswellic acids, curcumin, quercetin and resveratrol. These examples have in common that the achieved plasma concentrations were found to be often far below the determined IC50 values in vitro. On the other hand demonstrated therapeutic effects suggest a necessity of rethinking our pharmacokinetic understanding. In this light this review discusses the value of plasma levels as pharmacokinetic surrogates in comparison to the more informative value of tissue concentrations. Furthermore the need for new methodological approaches is addressed like the application of combinatorial approaches for identifying and pharmacokinetic investigations of active multi-components. Also the physiological relevance of exemplary in vitro assays and absorption studies in cell-line based models is discussed. All these topics should be ideally considered to avoid inaccurate predictions for the efficacy of herbal components in vivo and to unlock the “black box” of herbal mixtures.
An approach for the comparison of pair distribution functions (PDFs) has been developed using a similarity measure based on cross-correlation functions. The PDF is very sensitive to changes in the local structure, i.e. small deviations in the structure can cause large signal shifts and significant discrepancies between the PDFs. Therefore, a comparison based on pointwise differences (e.g. R values and difference curves) may lead to the assumption that the investigated PDFs as well as the corresponding structural models are not in agreement at all, whereas a careful visual inspection of the investigated structural models and corresponding PDFs may reveal a relatively good match. To quantify the agreement of different PDFs for those cases an alternative approach is introduced: the similarity measure based on cross-correlation functions. In this paper, the power of this application of the similarity measure to the analysis of PDFs is highlighted. The similarity measure is compared with the classical Rwp values as representative of the comparison based on pointwise differences as well as with the Pearson product-moment correlation coefficient, using polymorph IV of barbituric acid as an example.
Dimerization of Taspase1 activates an intrinsic serine protease function that leads to the catalytic Thr234 residue, which allows to catalyze the consensus sequence Q−3X−2D−1⋅G1X2D3D4, present in Trithorax family members and TFIIA. Noteworthy, Taspase1 performs only a single hydrolytic step on substrate proteins, which makes it impossible to screen for inhibitors in a classical screening approach. Here, we report the development of an HTRF reporter assay that allowed the identification of an inhibitor, Closantel sodium, that inhibits Taspase1 in a noncovalent fashion (IC50 = 1.6 μM). The novel inhibitor interferes with the dimerization step and/or the intrinsic serine protease function of the proenzyme. Of interest, Taspase1 is required to activate the oncogenic functions of the leukemogenic AF4-MLL fusion protein and was shown in several studies to be overexpressed in many solid tumors. Therefore, the inhibitor may be useful for further validation of Taspase1 as a target for cancer therapy.
Mit 71 Millionen chronisch erkrankten Patienten im Jahr 2015 stellt die chronische Hepatitis C-Virusinfektion eine wichtige Ursache für Zirrhose, Leberdekompensation und Leberkrebs dar.
Eine grundlegende Eigenschaft des Hepatitis C-Virus (HCV) ist die Biogenese modifizierter intrazellulärer Membranen. Das dabei aus dem endoplasmatischen Reticulum (ER) gebildete, sogenannte membranöse Netz (MW, membranous web) dient im Rahmen des HCV-Lebenszyklus als Gerüst für die Assemblierung eines Multi-Protein-Replikase-Komplexes. Das MW wird durch virale nicht-strukturelle Proteine wie NS5A induziert.
Das Multidomänen-Metalloprotein NS5A ist über seine verschiedenen Domänen sowohl bei der Replikation am MW als auch bei der viralen Assemblierung und Freisetzung in der Nähe von Lipidtropfen (LD, lipid droplet) maßgeblich beteiligt. Seine N-terminale amphipathische Helix (AH) spielt dabei über die vermittelte Assoziation von NS5A mit Membranen eine wichtige Rolle. Damit verbundene spezifische Lipidinteraktionen von NS5A unterliegen molekularen Umstrukturierungen, die benötigt werden, um NS5A für seine
verschiedenen Aufgaben im viralen Lebenszyklus anzupassen. Es liegen zwar Röntgen-strukturmodelle von Domäne 1-Dimeren und NMR-Strukturen zur AH vor, allerdings keine experimentellen Strukturen des NS5A-Proteins vollständiger Länge (NS5A fl) in seinem natürlichen Lipidmilieu. Trotz der essentiellen Bedeutung von NS5A für den HCV-Lebens-zyklus und langjähriger Forschung ist bisher nur wenig zur molekularen Funktionsweise von NS5A bekannt.
Dennoch konnten durch Screening hochpotente NS5A-Inhibitoren entdeckt und weiterentwickelt werden. NS5A-Inhibitoren tragen als direkt wirkende antivirale Arzneimittel(DAA, direct-acting antiviral) entscheidend zum Therapieerfolg bei der Behandlung der Hepatitis C bei. Trotz ihrer Bedeutung in der Therapie und intensiver Forschung ist der Wirk-mechanismus von NS5A-Inhibitoren bisher ungeklärt. Eine durch NS5A-Inhibitoren
induzierte intrazelluläre Umverteilung von NS5A und das ausschließliche Auftreten von Resistenz-assoziierten Mutationen (RAM) nahe der NS5A-Lipid-Interaktionsbereiche weisen jedoch auf einen Effekt der Inhibitoren auf die Lipid-NS5A-Interaktion hin.
Als grundlegende Hypothese dieser Arbeit wurde somit vermutet, dass abhängig vom NS5A umgebenden Lipidmilieu (MW oder LD) spezifische Lipid-Protein-Interaktionen Einfluss auf die Struktur und Funktion von NS5A nehmen und NS5A-Inhibitoren über eine Inhibition dieser Interaktionen wirken. Polyphosphoinositide (PPI) könnten dabei als Lipidinteraktionspartner eine besondere Rolle spielen, da sie bedeutend für die Membran-Kennzeichnung verschiedener Zellkompartimente sind und auch die Funktion von Membranproteinen regulieren können. Eine Interaktion von NS5A mit PtdIns(4,5)P2 wurde bereits publiziert.
Um basierend auf der postulierten Hypothese die mechanistischen Details im Wechselspiel von NS5A und intrazellulären Membranen sowie den dabei möglichen Effekt von
NS5A-Inhibitoren zu untersuchen, musste zunächst NS5A in ausreichender Menge, Reinheit und Qualität rekombinant hergestellt werden. Hierfür wurde ein entsprechendes Protokoll zur Proteinexpression durch Baculovirus-vermittelte Expression in Sf9-Insektenzellen und Strep-Tactin-Reinigung für das full length Protein und trunkierte Varianten etabliert.
In Kooperation mit einem Partner konnte unter Verwendung von giant unilamellar vesicles (GUVs) und konfokaler Mikroskopie gezeigt werden, dass unser full length Protein die Struktur von Membranen verändert (Membran-Remodellierung).
Die Stabilität des gereinigten Proteins und damit Effekte auf die Proteinfaltung wurden mittels Thermal shift assay (TSA) untersucht und dabei auch Effekte des NS5A-Inhibitors
Daclatasvir (DCV) und des Metall-Chelators EDTA überprüft. Die Bindung des Inhibitors hatte einen stabilisierenden Effekt auf die Proteinstruktur zur Folge.
Potentielle Interaktionsmuster mit Membranlipiden wurden mit Hilfe eines Protein lipid overlay assays (PLOA) detektiert. Zusätzlich zum in der Literatur bereits beschriebenen
Interaktionspartner PtdIns(4,5)P2 konnten weitere Lipid-Bindungspartner für NS5A identifiziert werden. Dabei legen die gewonnenen Daten nahe, dass die Interaktion über die Domäne 1 von NS5A vermittelt wird, wobei die Domänen 2 und 3 die Affinität zu den Lipidbindungspartnern erhöht, aber nicht das Phospholipid-Bindungsmuster verändert.
DCV hatte im PLOA keine qualitativen Auswirkungen auf das Lipid-Bindungsmuster. Die Lipidinteraktionen wurden mittels eines Liposomen-Rekonstitutionsmodells validiert.
In silico konnten basierend auf verfügbaren, experimentellen Strukturdaten und einem dynamischen Modell drei Cluster basischer Aminosäuren in NS5A-D1-AH als mögliche
PPI-Bindungsstellen identifiziert werden. Basierend auf dem Strukturmodell wurde eine Mutationsstrategie zur Charakterisierung der potentiellen PPI-Bindungsstellen entwickelt.
...
Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic β-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1/TM2 groove region. The variants were tested in E. coli ΔacrBΔacrD against β-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic β-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.
Cyclic GMP (cGMP) is a second messenger that regulates numerous physiological and pathophysiological processes. In recent years, more and more studies have uncovered multiple roles of cGMP signalling pathways in the somatosensory system. Accumulating evidence suggests that cGMP regulates different cellular processes from embryonic development through to adulthood. During embryonic development, a cGMP-dependent signalling cascade in the trunk sensory system is essential for axon bifurcation, a specific form of branching of somatosensory axons. In adulthood, various cGMP signalling pathways in distinct cell populations of sensory neurons and dorsal horn neurons in the spinal cord play an important role in the processing of pain and itch. Some of the involved enzymes might serve as a target for future therapies. In this review, we summarise the knowledge regarding cGMP-dependent signalling pathways in dorsal root ganglia and the spinal cord during embryonic development and adulthood, and the potential of targeting these pathways.
LINKED ARTICLES
This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc
The intriguing (μ-hydrido)diboranes(4) with their prominent pristine representative [B2H5]− have mainly been studied theoretically. We now describe the behavior of the planarized tetraaryl (μ-hydrido)diborane(4) anion [1H]− in cycloaddition reactions with the homologous series of heterocumulenes CO2, iPrNCO, and iPrNCNiPr. We show that a C=O bond of CO2 selectively activates the B−B bond of [1H]−, while the μ-H ligand is left untouched ([2H]−). The carbodiimide iPrNCNiPr, in contrast, neglects the B−B bond and rather adds the B-bonded H− ion to its central C atom to generate a formamidinate bridge across the B2 pair ([3]−). As a hybrid, the isocyanate iPrNCO combines the reactivity patterns of both its congeners and gives two products: one of them ([4H]−) is related to [2H]−, the other ([5]−) is an analog of [3]−. We finally propose a mechanistic scenario that rationalizes the individual reaction outcomes and combines them to a coherent picture of B–B vs. B–H bond activation.
Ginger (Zingiber officinale Roscoe) is widely used as medicinal plant. According to the Committee on Herbal Medicinal Products (HMPC), dried powdered ginger rhizome can be applied for the prevention of nausea and vomiting in motion sickness (well-established use). Beyond this, a plethora of pre-clinical studies demonstrated anti-cancer, anti-oxidative, or anti-inflammatory actions. 6-Shogaol is formed from 6-gingerol by dehydration and represents one of the main bioactive principles in dried ginger rhizomes. 6-Shogaol is characterized by a Michael acceptor moiety being reactive with nucleophiles. This review intends to compile important findings on the actions of 6-shogaol as an anti-inflammatory compound: in vivo, 6-shogaol inhibited leukocyte infiltration into inflamed tissue accompanied with reduction of edema swelling. In vitro and in vivo, 6-shogaol reduced inflammatory mediator systems such as COX-2 or iNOS, affected NFκB and MAPK signaling, and increased levels of cytoprotective HO-1. Interestingly, certain in vitro studies provided deeper mechanistic insights demonstrating the involvement of PPAR-γ, JNK/Nrf2, p38/HO-1, and NFκB in the anti-inflammatory actions of the compound. Although these studies provide promising evidence that 6-shogaol can be classified as an anti-inflammatory substance, the exact mechanism of action remains to be elucidated. Moreover, conclusive clinical data for anti-inflammatory actions of 6-shogaol are largely lacking.
Chemical language models enable de novo drug design without the requirement for explicit molecular construction rules. While such models have been applied to generate novel compounds with desired bioactivity, the actual prioritization and selection of the most promising computational designs remains challenging. Herein, we leveraged the probabilities learnt by chemical language models with the beam search algorithm as a model-intrinsic technique for automated molecule design and scoring. Prospective application of this method yielded novel inverse agonists of retinoic acid receptor-related orphan receptors (RORs). Each design was synthesizable in three reaction steps and presented low-micromolar to nanomolar potency towards RORγ. This model-intrinsic sampling technique eliminates the strict need for external compound scoring functions, thereby further extending the applicability of generative artificial intelligence to data-driven drug discovery.
Pancreatic cancer is a common malignant tumor with a high incidence and mortality rate. The prognosis of patients with pancreatic cancer is considerably poor due to the lack of effective treatment in clinically. Despite numerous studies have revealed that baicalein, a natural product, is responsible for suppressing multiple cancer cells proliferation, motility and invasion. The mechanism by which baicalein restraining pancreatic cancer progression remains unclear. In this study, we firstly verified that baicalein plays a critical role in inhibiting pancreatic tumorigenesis in vitro and in vivo. Then we analyzed the alteration of microRNAs (miRNAs) expression levels in Panc-1 cells incubated with DMSO, 50 and 100 μM baicalein by High-Throughput sequencing. Intriguingly, we observed that 20 and 39 miRNAs were accordingly up- and down-regulated through comparing Panc-1 cells exposed to 100 μM baicalein with the control group. Quantitative PCR analysis confirmed that miR-139-3p was the most up-regulated miRNA after baicalein treatment, while miR-196b-5p was the most down-regulated miRNA. Further studies showed that miR-139-3p induced, miR-196b-5p inhibited the apoptosis of Panc-1 cells via targeting NOB1 and ING5 respectively. In conclusion, we demonstrated that baicalein is a potent inhibitor against pancreatic cancer by modulating the expression of miR-139-3p or miR-196b-5p.
Introns of human transfer RNA precursors (pre-tRNAs) are excised by the tRNA splicing endonuclease TSEN in complex with the RNA kinase CLP1. Mutations in TSEN/CLP1 occur in patients with pontocerebellar hypoplasia (PCH), however, their role in the disease is unclear. Here, we show that intron excision is catalyzed by tetrameric TSEN assembled from inactive heterodimers independently of CLP1. Splice site recognition involves the mature domain and the anticodon-intron base pair of pre-tRNAs. The 2.1-Å resolution X-ray crystal structure of a TSEN15–34 heterodimer and differential scanning fluorimetry analyses show that PCH mutations cause thermal destabilization. While endonuclease activity in recombinant mutant TSEN is unaltered, we observe assembly defects and reduced pre-tRNA cleavage activity resulting in an imbalanced pre-tRNA pool in PCH patient-derived fibroblasts. Our work defines the molecular principles of intron excision in humans and provides evidence that modulation of TSEN stability may contribute to PCH phenotypes.
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.
Extracts of frankincense, the gum resin of Boswellia species, have been extensively used in traditional folk medicine since ancient times and are still of great interest as promising anti-inflammatory remedies in Western countries. Despite their common therapeutic use and the intensive pharmacological research including studies on active ingredients, modes of action, bioavailability, pharmacokinetics, and clinical efficacy, frankincense preparations are available as nutraceuticals but have not yet approved as a drug on the market. A major issue of commercially available frankincense nutraceuticals is the striking differences in their composition and quality, especially related to the content of boswellic acids (BAs) as active ingredients, mainly due to the use of material from divergent Boswellia species but also because of different work-up and extraction procedures. Here, we assessed three frequently used frankincense-based preparations for their BA content and the interference with prominent pro-inflammatory actions and targets that have been proposed, that is, 5-lipoxygenase and leukotriene formation in human neutrophils, microsomal prostaglandin E2 synthase-1, and inflammatory cytokine secretion in human blood monocytes. Our data reveal striking differences in the pharmacological efficiencies of these preparations in inflammation-related bioassays which obviously correlate with the amounts of BAs they contain. In summary, high-quality frankincense extracts display powerful anti-inflammatory effectiveness against multiple targets which can be traced back to BAs as bioactive ingredients.
Currently, a wide variety of complex non-oral dosage forms are entering the global healthcare market. Although many assays have been described in recent research, harmonized procedures and standards for testing their in vitro performance remain widely unexplored. Among others, dialysis-based techniques such as the Pharma Test Dispersion Releaser are developed for testing the release of drugs from nanoparticles, liposomes, or extracellular vesicle preparations. Here, we provide advanced strategies and practical advice for the development and validation of dialysis-based techniques, including documentation, analysis, and interpretation of the raw data. For this purpose, key parameters of the release assay, including the hydrodynamics in the device at different stirring rates, the selectivity for particles and molecules, as well as the effect of excipients on drug permeation were investigated. At the highest stirring rate, a more than twofold increase in the membrane permeation rate (from 0.99 × 10−3 to 2.17 × 10−3 cm2/h) was observed. Additionally, we designed a novel computer model to identify important quality parameters of the dialysis experiment and to calculate error-corrected release profiles. Two hydrophilic creams of diclofenac, Voltaren® Emulgel, and Olfen® gel, were tested and provide first-hand evidence of the robustness of the assay in the presence of semisolid dosage forms.
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.
NMR structure calculation using NOE-derived distance restraints requires a considerable number of assignments of both backbone and sidechains resonances, often difficult or impossible to get for large or complex proteins. Pseudocontact shifts (PCSs) also play a well-established role in NMR protein structure calculation, usually to augment existing structural, mostly NOE-derived, information. Existing refinement protocols using PCSs usually either require a sizeable number of sidechain assignments or are complemented by other experimental restraints. Here, we present an automated iterative procedure to perform backbone protein structure refinements requiring only a limited amount of backbone amide PCSs. Already known structural features from a starting homology model, in this case modules of repeat proteins, are framed into a scaffold that is subsequently refined by experimental PCSs. The method produces reliable indicators that can be monitored to judge about the performance. We applied it to a system in which sidechain assignments are hardly possible, designed Armadillo repeat proteins (dArmRPs), and we calculated the solution NMR structure of YM4A, a dArmRP containing four sequence-identical internal modules, obtaining high convergence to a single structure. We suggest that this approach is particularly useful when approximate folds are known from other techniques, such as X-ray crystallography, while avoiding inherent artefacts due to, for instance, crystal packing.
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.
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.
Background and Purpose: Activation of hepatic thyroid hormone receptor β (THR-β) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-β agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-β agonist, as a prototype to investigate the effects of THR-β activation in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis.
Experimental Approach: C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg−1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-β activation.
Key Results: Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis.
Conclusion and Implications: Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity.
Computational oral absorption models, in particular PBBM models, provide a powerful tool for researchers and pharmaceutical scientists in drug discovery and formulation development, as they mimic and can describe the physiologically processes relevant to the oral absorption. PBBM models provide in vivo context to in vitro data experiments and allow for a dynamic understanding of in vivo drug disposition that is not typically provided by data from standard in vitro assays. Investigations using these models permit informed decision-making, especially regarding to formulation strategies in drug development. PBBM models, but can also be used to investigate and provide insight into mechanisms responsible for complex phenomena such as food effect in drug absorption. Although there are obviously still some gaps regarding the in silico construction of the gastrointestinal environment, ongoing research in the area of oral drug absorption (e.g. the UNGAP, AGE-POP and InPharma projects) will increase knowledge and enable improvement of these models.
PBBM can nowadays provide an alternative approach to the development of in vitro–in vivo correlations. The case studies presented in this thesis demonstrate how PBBM can address a mechanistic understanding of the negative food effect and be used to set clinically relevant dissolution specification for zolpidem immediate release tablets. In both cases, we demonstrated the importance of integrating drug properties with physiological variables to mechanistically understand and observe the impact of these parameters on oral drug absorption.
Various complex physiological processes are initiated upon food consumption, which can enhance or reduce a drug’s dissolution, solubility, and permeability and thus lead to changes in drug absorption. With improvements in modeling and simulation software and design of in vitro studies, PBBM modeling of food effects may eventually serve as a surrogate for clinical food effect studies for new doses and formulations or drugs. Furthermore, the application of these models may be even more critical in case of compounds where execution of clinical studies in healthy volunteers would be difficult (e.g., oncology drugs).
In the fourth chapter we have demonstrated the establishment of the link between biopredictive in vitro dissolution testing (QC or biorelevant method) PBBM coupled with PD modeling opens the opportunity to set truly clinically relevant specifications for drug release. This approach can be extended to other drugs regardless of its classification according to the BCS.
With the increased adoption of PBBM, we expect that best practices in development and verification of these models will be established that can eventually inform a regulatory guidance. Therefore, the application of Physiologically Based Biopharmaceutical Modelling is an area with great potential to streamline late-stage drug development and impact on regulatory approval procedures.
Polo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.
Organ-on-a-chip technology has the potential to accelerate pharmaceutical drug development, improve the clinical translation of basic research, and provide personalized intervention strategies. In the last decade, big pharma has engaged in many academic research cooperations to develop organ-on-a-chip systems for future drug discoveries. Although most organ-on-a-chip systems present proof-of-concept studies, miniaturized organ systems still need to demonstrate translational relevance and predictive power in clinical and pharmaceutical settings. This review explores whether microfluidic technology succeeded in paving the way for developing physiologically relevant human in vitro models for pharmacology and toxicology in biomedical research within the last decade. Individual organ-on-a-chip systems are discussed, focusing on relevant applications and highlighting their ability to tackle current challenges in pharmacological research.
Two subvalent, redox-active diborane(4) anions, [3]4− and [3]2−, carrying exceptionally high negative charge densities are reported: Reduction of 9-methoxy-9-borafluorene with Li granules without stirring leads to the crystallization of the B(sp3)−B(sp2) diborane(5) anion salt Li[5]. [5]− contains a 2,2′-biphenyldiyl-bridged B−B core, a chelating 2,2′-biphenyldiyl moiety, and a MeO substituent. Reduction of Li[5] with Na metal gives the Na+ salt of the tetraanion [3]4− in which two doubly reduced 9-borafluorenyl fragments are linked via a B−B single bond. Comproportionation of Li[5] and Na4[3] quantitatively furnishes the diborane(4) dianion salt Na2[3], the doubly boron-doped congener of 9,9′-bis(fluorenylidene). Under acid catalysis, Na2[3] undergoes a formal Stone–Wales rearrangement to yield a dibenzo[g,p]chrysene derivative with B=B core. Na2[3] shows boron-centered nucleophilicity toward n-butyl chloride. Na4[3] produces bright blue chemiluminescence when exposed to air.
Although overexpression and hyperactivity of protein kinases are causative for a wide range of human cancers, protein kinase inhibitors currently approved as cancer drugs address only a limited number of these enzymes. To identify new chemotypes addressing alternative protein kinases, the basic structure of a known PLK1/VEGF-R2 inhibitor class was formally dissected and reassembled. The resulting 7-(2-anilinopyrimidin-4-yl)-1-benzazepin-2-ones were synthesized and proved to be dual inhibitors of Aurora A kinase and VEGF receptor kinases. Crystal structures of two representatives of the new chemotype in complex with Aurora A showed the ligand orientation in the ATP binding pocket and provided the basis for rational structural modifications. Congeners with attached sulfamide substituents retained Aurora A inhibitory activity. In vitro screening of two members of the new kinase inhibitor family against the cancer cell line panel of the National Cancer Institute (NCI) showed antiproliferative activity in the single-digit micromolar concentration range in the majority of the cell lines.
The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5′-untranslated region (5′-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5′-UUUCGU-3′ hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1H, 13C and 15N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
Die Fähigkeit der spezifischen und kontextabhängigen zellulären Adaption auf intrinsische und/oder extrinsische Signale ist das Fundament zellulärer Homöostase. Verschiedene Signale werden von Membranrezeptoren oder intrazellulären Rezeptoren erkannt und ermöglichen die molekulare Anpassung zellulärer Prozesse. Komplexe, ineinandergreifende Proteinnetzwerke sind dabei elementar in der Regulation der Zelle. Proteine und deren Funktionen werden dabei nach Bedarf reguliert und unterliegen einem ständigen proteolytischen Umsatz.
Die stimulusabhängige Gentranskription und/oder Proteintranslation nimmt hier eine zentrale Stellung ein, da die zugrundeliegende Maschinerie die Komposition und Funktion der Proteinnetzwerke entsprechend anpassen kann. Zusätzlich zur Regulation der Proteinabundanz werden Proteine posttranslational modifiziert, um deren Eigenschaften rasch zu ändern. Zu posttranslationalen Modifikationen zählen die Ubiquitinierung und/oder Phosphorylierung, welche die Proteinfunktionen hochdynamisch regulieren. Deregulierte Proteinnetzwerke werden oft mit Neurodegeneration und Autoimmun- oder Krebserkrankungen assoziiert. Auch Infektionen mit humanpathogenen Bakterien greifen stark in den Regulierungsprozess von Proteinnetzwerken und deren Funktionen ein. Die zelluläre Homöostase wird dadurch herausgefordert.
Bakterien der Gattung Salmonella sind zoonotische, gramnegative, fakultativ intrazelluläre Pathogene, welche weltweit millionenfach Salmonellen-erkrankungen hervorrufen. Von besonderer Bedeutung ist dabei Salmonella enterica serovar Typhimurium (hiernach Salmonella), welches im Menschen, meist durch mangelnde Hygienemaßnahmen, Gastroenteritis auslöst.
Immunität in Epithelzellen wird über das angeborene Immunsystem vermittelt und dient der Pathogenerkennung und -bekämpfung. Die Toll-like Rezeptoren (TLR) gehören zu den Mustererkennungsrezeptoren (pattern recognition receptors), welche spezifische mikrobielle Strukturen detektieren und eine kontextabhängige zelluläre Antwort generieren. Danger-Rezeptoren erkennen hingegen nicht direkt das Pathogen, sondern zelluläre Perturbationen, welche durch Zellschäden oder bakterielle Invasionen verursacht werden. Die intrinsische Fähigkeit der Wirtszelle, sich gegen Infektionen/Gefahren zu wehren wird dabei als zellautonome Immunität bezeichnet. Dabei nehmen induzierte proinflammatorische Signalwege und zelluläre Stressantworten eine wichtige Stellung ein. Die zelluläre Stressantwort aktiviert unter anderem die selektive Autophagie. Diese kann spezifisch aberrante Organelle, Proteine und invasive Pathogene abbauen. Ein weiterer Stresssignalweg ist die integrated stress response (ISR), welche eine selektive Proteintranslation erlaubt und damit die Auflösung des proteintoxischen Stresses ermöglicht.
Zur Penetration von Epithelzellen benötigt Salmonella ein komplexes System an Virulenzfaktoren, welches die bakterielle Internalisierung und Proliferation in der Wirtszelle ermöglicht. Salmonella nutzt dazu ein Typ-III-Sekretionssystem. Das System sekretiert bakterielle Virulenzfaktoren in die Zelle, sodass eine hochspezifische Modulierung des Wirtes erzwungen wird.
Die Virulenzfaktoren SopE und SopE2 spielen dabei eine Schlüsselrolle, da sie die Pathogenität von Salmonella maßgeblich vermitteln. Durch molekulare Mimikry von Wirts GTP (Guanosintriphosphat) -Austauschfaktoren aktivieren SopE und SopE2 die Rho GTPasen CDC42 und Rac1. GTP-geladenes CDC42 und Rac1 wiederum aktivieren das Aktinzytoskelett und stimulieren die Polymerisierung von Aktinfilamenten über den Arp2/3-Komplex an der Invasionsstelle. Das Pathogen wird dadurch in ein membranumhülltes Vesikel, die sogenannte Salmonella-containing Vakuole (SCV), aufgenommen. Die SCV stellt eine protektive, replikative, intrazelluläre Nische des Pathogens dar und wird permanent durch verschiedene Virulenzfaktoren moduliert.
Im Allgemeinen führt die Aktivierung von Mustererkennungsrezeptoren und Danger-Rezeptoren also zu einer zellulären Stressantwort und Entzündungsreaktion, wodurch es zur Bekämpfung der Infektion kommt. Inflammatorische Signalwege werden meist über den zentralen Transkriptionsfaktor NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) vermittelt. NF-κB bewirkt die Induktion von proinflammatorischen Effektoren und Stressgenen. Zellautonome Immunität wird zusätzlich durch antibakterielle Autophagie ermöglicht, wobei Salmonella selektiv über das lysosomale System abgebaut werden. Das bakterielle Typ-III-Sekretionssystem verursacht an einigen wenigen SCVs Membranschäden, sodass Salmonella das Wirtszytosol penetrieren. Zytosolische Bakterien werden dabei spezifisch ubiquitiniert. Dies erlaubt die Erkennung durch die Autophagie-Maschinerie.
In der vorliegenden Arbeit wurde die zellautonome Immunität von Epithelzellen während einer akuten Salmonella Infektion durch quantitative Proteomik untersucht...