Institutes
Refine
Year of publication
Document Type
- Doctoral Thesis (307)
- Article (278)
- Preprint (38)
- Contribution to a Periodical (25)
- Book (17)
- Report (2)
Language
- English (514)
- German (152)
- Multiple languages (1)
Has Fulltext
- yes (667)
Is part of the Bibliography
- no (667)
Keywords
- RNA (12)
- SARS-CoV-2 (10)
- NMR spectroscopy (9)
- inflammation (9)
- photochemistry (9)
- NMR (8)
- Biochemistry (7)
- Cell biology (7)
- E2 enzyme (6)
- TRACT (6)
Institute
- Biochemie, Chemie und Pharmazie (667)
- Präsidium (42)
- Medizin (35)
- Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) (30)
- Zentrum für Biomolekulare Magnetische Resonanz (BMRZ) (30)
- Biowissenschaften (23)
- MPI für Biophysik (15)
- Physik (12)
- Zentrum für Arzneimittelforschung, Entwicklung und Sicherheit (ZAFES) (5)
- Geowissenschaften / Geographie (4)
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotide-binding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.
Biogenesis of mitochondrial cytochrome c oxidase (COX) relies on a large number of assembly factors, among them the transmembrane protein Surf1. The loss of human Surf1 function is associated with Leigh syndrome, a fatal neurodegenerative disorder caused by severe COX deficiency. In the bacterium Paracoccus denitrificans, two homologous proteins, Surf1c and Surf1q, were identified, which we characterize in the present study. When coexpressed in Escherichia coli together with enzymes for heme a synthesis, the bacterial Surf1 proteins bind heme a in vivo. Using redox difference spectroscopy and isothermal titration calorimetry, the binding of the heme cofactor to purified apo-Surf1c and apo-Surf1q is quantified: Each of the Paracoccus proteins binds heme a in a 1:1 stoichiometry and with Kd values in the submicromolar range. In addition, we identify a conserved histidine as a residue crucial for heme binding. Contrary to most earlier concepts, these data support a direct role of Surf1 in heme a cofactor insertion into COX subunit I by providing a protein-bound heme a pool.
Biological membranes are complex and dynamic assemblies of lipids and proteins. Poikilothermic organisms including bacteria, fungi, reptiles, and fish do not control their body temperature and must adapt their membrane lipid composition in order to maintain membrane fluidity in the cold. This adaptive response was termed homeoviscous adaptation and has been frequently studied with a specific focus on the acyl chain composition of membrane lipids. Mass spectrometry-based lipidomics can nowadays provide more comprehensive insights into the complexity of lipid remodeling during adaptive responses. Eukaryotic cells compartmentalize biochemical processes in organelles with characteristic surface properties, and the lipid composition of organelle membranes must be tightly controlled in order to maintain organelle function and identity during adaptive responses. Some highly differentiated cells such as neurons maintain unique lipid compositions with specific physicochemical properties. To date little is known about the sensory mechanisms regulating the acyl chain profile in such specialized cells or during adaptive responses. Here we summarize our current understanding of lipid metabolic networks with a specific focus on the role of physicochemical membrane properties for the regulation of the acyl chain profile during homeoviscous adaptation. By comparing the mechanisms of the bacterial membrane sensors with the prototypical eukaryotic lipid packing sensor Mga2 from Saccharomyces cerevisiae, we identify common operational principles that might guide our search for novel membrane sensors in different organelles, organisms, and highly specialized cells.
We demonstrated previously that 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, can be phosphorylated by p38 MAPK-regulated MAPKAP kinases (MKs). Here we show that mutation of Ser-271 to Ala in 5-LO abolished MK2 catalyzed phosphorylation and clearly reduced phosphorylation by kinases prepared from stimulated polymorphonuclear leukocytes and Mono Mac 6 cells. Compared with heat shock protein 27 (Hsp-27), 5-LO was a weak substrate for MK2. However, the addition of unsaturated fatty acids (i.e. arachidonate 1-50 microm) up-regulated phosphorylation of 5-LO, but not of Hsp-27, by active MK2 in vitro, resulting in a similar phosphorylation as for Hsp-27. 5-LO was phosphorylated also by other serine/threonine kinases recognizing the motif Arg-Xaa-Xaa-Ser (protein kinase A, Ca(2+)/calmodulin-dependent kinase II), but these activities were not increased by fatty acids. HeLa cells expressing wild type 5-LO or S271A-5-LO, showed prominent 5-LO activity when incubated with Ca(2+)-ionophore plus arachidonate. However, when stimulated with only exogenous arachidonic acid, activity for the S271A mutant was significantly lower as compared with wild type 5-LO. It appears that phosphorylation at Ser-271 is more important for 5-LO activity induced by a stimulus that does not prominently increase intracellular Ca(2+) and that arachidonic acid stimulates leukotriene biosynthesis also by promoting this MK2-catalyzed phosphorylation.
Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.
Epigenetic control of microsomal prostaglandin E synthase-1 by HDAC-mediated recruitment of p300
(2017)
Nonsteroidal anti-inflammatory drugs are the most widely used medicine to treat pain and inflammation, and to inhibit platelet function. Understanding the expression regulation of enzymes of the prostanoid pathway is of great medical relevance. Histone acetylation crucially controls gene expression. We set out to identify the impact of histone deacetylases (HDACs) on the generation of prostanoids and examine the consequences on vascular function. HDAC inhibition (HDACi) with the pan-HDAC inhibitor, vorinostat, attenuated prostaglandin (PG)E2 generation in the murine vasculature and in human vascular smooth muscle cells. In line with this, the expression of the key enzyme for PGE2 synthesis, microsomal PGE synthase-1 (PTGES1), was reduced by HDACi. Accordingly, the relaxation to arachidonic acid was decreased after ex vivo incubation of murine vessels with HDACi. To identify the underlying mechanism, chromatin immunoprecipitation (ChIP) and ChIP-sequencing analysis were performed. These results suggest that HDACs are involved in the recruitment of the transcriptional activator p300 to the PTGES1 gene and that HDACi prevented this effect. In line with the acetyltransferase activity of p300, H3K27 acetylation was reduced after HDACi and resulted in the formation of heterochromatin in the PTGES1 gene. In conclusion, HDAC activity maintains PTGES1 expression by recruiting p300 to its gene.
Der Fokus der Arbeit liegt auf der Untersuchung von Wechselwirkungen zwischen Molekülen in selbst-anordnenden Monolagen (SAMs) auf Goldoberflächen mittels Rastertunnelmikroskopie und komplementären Methoden wie z.B. Infrarot-Reflektions-Absorptions-Spektro-skopie.
In dieser Arbeit wurde das kürzlich etablierte Konzept von eingebetteten Dipolmomenten in aromatischen, SAM-bildenden Molekülen eingehender untersucht. Das Ausmaß des Dipol-moments und die Größe der SAM-bildenden Moleküle wurden synthetisch variiert und der Einfluss auf die Struktur und elektronischen Eigenschaften der SAMs untersucht. Binäre, gemischte Monolagen aus SAM-bildenden Molekülen mit "entgegen gerichteten", Dipolmomenten wurden hergestellt und charakterisiert. Zur Herstellung der binären, gemischten Monolagen wurden zwei Methoden verwendet: die Monolagen wurden a) aus bereits gemischten Lösungen der Moleküle abgeschieden oder b) eine reine SAM in die Lösung des anderen Moleküls eingelegt, so dass ein Austausch stattfand. Der Vergleich der beiden Methoden ermöglicht Rückschlüsse über die Abscheidungsprozesse. Die Charakterisierung der SAMs dieser Mischungsreihen gab Aufschluss über Eigenschaften wie Packungsdichte, Austrittsarbeit, elektronischen Ladungstransport in Monolagen und Orientierung der Moleküle relativ zur Oberfläche und erlaubte Schlussfolgerungen über die Mischbarkeit und das Ausmaß der Dipolwechselwirkungen der Moleküle in der Monolage. In einem ähnlichen Ansatz zu dem oben beschriebenen Vorgehen wurden Quadrupolwechselwirkungen zwischen SAM-bildenen, Benzol-, Naphtalin- und Anthracenderivaten untersucht. In Mischungsreihen wurden SAMs von nicht- und teilweise (hoch)fluorierten, SAM-bildenden Molekülen auf Goldoberflächen charakterisiert. Die Ergebnisse der Untersuchungen können bei der gezielten Einstellung der elektronischen Eigenschaften in elektronischen Bauteilen wie OFETs Anwendung finden.
In einem weiteren Projekt wurde der Einfluss von polaren Endgruppen auf die in situ Abspaltung von Schutzgruppen an Terphenylthiol-Derivaten untersucht, wobei die Ergebnisse zum Aufbau größerer, aus organischer Elektronik bestehender, Netzwerke verwendet werden können.
The role of USP22 in nucleic acid sensing pathways and interferon-induced necroptotic cell death
(2023)
Every day, living organisms are challenged by internal and external factors that threaten to bring imbalance to their tightly regulated systems and disrupt homeostasis, leading to degeneration, and ultimately death. More than ever, we face the challenge of combating diseases such as COVID-19 caused by infection with the SARS-CoV-2 coronavirus. It is therefore crucial to identify host factors that control antiviral defense mechanisms. In addition, in the fight against cancer, it is becoming increasingly important to identify markers that could be used for targeted therapy to influence cellular processes and determine cell fate.
As a deubiquitylating enzyme, ubiquitin specific peptidase 22 (USP22) mediates the removal of the small molecule ubiquitin, which is post-translationally added to target proteins, thereby regulating several important processes such as protein degradation, activation or localization. Through its deubiquitylating function, USP22 controls several biological processes such as cell cycle regulation, proliferation and cancer immunoresistance by modulating key proteins involved in these pathways. Lately, USP22 was reported to positively regulate TNFα-mediated necroptosis, an inflammatory type of programmed cell death, in various human tumor cell lines by affecting RIPK3 phosphorylation. In addition, USP22 as a part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) transcription complex is known to regulate gene expression by removing ubiquitin from histones H2A and H2B. However, little is known about the role of USP22 in global gene expression.
In this study, we performed a genome-wide screen in the human colon carcinoma cell line HT-29 and identified USP22 as a key negative regulator of basal interferon (IFN) expression. We further demonstrated that the absence of USP22 results in increased STING activity and ubiquitylation, both basally and in response to stimulation with the STING agonist 2'3'-cGAMP, thereby affecting IFNλ1 expression and basal expression of antiviral ISGs. In addition, we were able to establish USP22 as a critical host factor in controlling SARS-CoV-2 infection by regulating infection, replication, and the generation of infectious virus particles, which we attribute in part to its role in regulating STING signaling.
In the second part of the study, we connected the findings of USP22-dependent regulation of IFN signaling and TNFα-induced necroptosis and investigated the role of USP22 during necroptosis induced by the synergistic action of IFN and the Smac mimetic BV6 in caspase-deficient settings. We identified USP22 as a negative regulator of IFN-induced necroptosis, which does not depend on STING expression, but relies on a yet unknown mechanism.
In summary, we identify USP22 as an important regulator of IFN signaling with important implications for the defense against viral infections and regulation of the necroptotic pathway that could be exploited for devising targeted therapeutic strategies against viral infections and related diseases like COVID-19, and advancing precision medicine in cancer treatment.
The accumulation and distribution of characteristic secondary products in the different organs of an Aloe plant (A. succotrina Lam.) were studied by high performance liquid chromatography for the first time. In the leaves of the Aloe plant, only anthrone-C-glycosyls of the 7-hydroxyaloin type and, for the first time in plant material, the free anthraquinone 7-hydroxyaloeemodin were found. In contrast to previous reports on the distribution of secondary products in Aloe plants, anthrone-C-glycosyls were also detected in flowers, bracts and the inflorescence axis of the species examined. Aloesaponol I, a tetrahydroanthracene aglycone, was only present in the underground organs and in the stem. The 2-alkylchromone-C-glucosyl aloeresin B showed no specific occurrence as it was found in every type of organ. Based on these results and the findings of recent studies on Aloe roots and flowers, a distribution scheme of polyketide types in the Aloe plant was established. It suggests a separate and independent anthranoid metabolism for underground Aloe organs and stem on the one hand, and for leaves and inflorescence organs on the other hand. In the latter structures anthranoid metabolism seems to be additionally compartmentalized as the anthranoid pro files of inflorescence organs and leaves differ in two points relevant to anthranoid biosynthe sis: firstly, the occurrence of anthrone aglycones and secondly, the individual content of corresponding anthrone-C-glucosyl diastereomers.
From the leaf exudate of Aloe lateritia ENGLER the C-glucosyl com pounds homonataloin, aloeresin A and aloesin (synon. aloeresin B) were isolated together with the anthraquinone nataloeem odin-8-methylether and spectroscopically identified. Hom onataloin, widely distributed in Aloe species, was separated into homonataloin A and B by combined TLC and DCCC. In their 1 D and 2D 1H NMR spectra only the shifts of the 2′-hydroxyl protons of both glucosyl residues differ significantly, indicative of 10 S (A) resp. 10 S (B) configurations. In both com pounds the anthrone is in β-position of the D-glucopyranosyl, as determined by the large coupling constants of the anomeric protons. The 13C NMR signals are unambiguously assigned by the use of DEPT, APT and gated-decoupling methods. Only the chemical shifts of C -11 and C -14 show significant differences between both diastereomers due to the adjacent 2′-sugar hydroxyls. The two homonataloins differ mostly in optical rotation and circulardichroism due to different configurations at C - 10 of the anthrone part. The absolute configurations of the diastereomers are determined by correlation of their CD spectra with the CD spectra of the structural analogues 7-hydroxyaloins A and B, which shows that hom onataloin A is the 10 S, 1′S-compound and that homonataloin B has 10 R, 1′S-configuration.
To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.
Today, the term buchu refers to the two species in commerce, Agathosma betulina (P.J.Bergius) Pillans and Agathosma crenulata (L.) Pillans (Rutaceae). Its traditional use in urinary tract infections and related ailments made it a popular remedy, specifically in the US, in 19th century, but with the advent of antibiotics it became largely obsolete. Recent focus is on technological use and on the essential oil for use in the perfume and food-flavouring industry. A review of the scarce pharmacological research revealed moderate antimicrobial activity for a leaf extract but not the essential oil of both species in the MIC assay. In the 5-lipoxygenase (5-LO) assay the essential oil of both species revealed IC50 values of 50.37 ± 1.87 μg/ml and 59.15 ± 7.44 μg/ml, respectively. In another study 98% inhibitory activity was determined for 250 μg/ml of an ethanolic extract of A. betulina on cyclooxygenase (COX)-1 and a 25% inhibitory activity on COX-2. Analgesic activity of an ethanolic extract of A. betulina was shown in mice. Moderate antioxidant activity was determined for methanol:dichlormethane extracts of A. betulina and A. crenulata and an aqueous extract of A. betulina showed a Trolox equivalent antioxidant capacity (TEAC) of 11.8 µM Trolox. Recent in vitro studies with a commercial aqueous extract of buchu revealed increased uptake of glucose added to 3T3-L1 cell line, significant inhibition of the respiratory burst of neutrophils and monocytes, reduction in the expression of adhesion molecules and inhibition of the release of IL-6 and TNF-α. In diabetic rats the ingestion of aqueous buchu extract completely normalized the glucose level and in rats receiving a high fat diet the consumption of aqueous buchu extract resulted in less weight gain and less intraperitoneal fat gain as well as reduction of elevated blood pressure to normal associated with cardioprotective effects. Limitations in the hitherto conducted research lie in the undisclosed composition of the buchu extracts used and the difficulty in extrapolating data from animal studies to humans. Health claims for buchu products need to be substantiated by randomized, double-blind and placebo-controlled studies. Only then can they be promoted for their true therapeutic potential.
Necroptosis is an immunogenic form of programmed cell death characterized by plasma membrane accumulation of activated mixed lineage kinase domain-like (MLKL) that eventually leads to membrane disruption and release of danger-associated molecular patterns (DAMPs). Necroptotic cell death is tightly controlled by checkpoints, including compartmentalization as well as post-translational modifications (PTMs), like phosphorylation and ubiquitination of receptor-interacting protein kinase (RIPK) 1, RIPK3 and MLKL. Removal of plasma membrane-located activated MLKL via endocytosis or exocytosis can counteract necroptosis, but up till now, the exact mechanisms by which necroptosis is regulated downstream of MLKL activation and oligomerization are not fully understood.
Ubiquitination is a key post-translational modification that regulates various cellular processes including cell survival and cell death signaling via ubiquitination of RIPK1, RIPK3 and MLKL. M1-linked (linear) poly-ubiquitination is mediated exclusively by the linear ubiquitin chain assembly complex (LUBAC) which critically regulates cell fate and immune signaling via death receptors such as TNF receptor 1 (TNFR1).
In this study, we demonstrate that M1 poly-Ubiquitin (poly-Ub) increases during necroptosis which can be blocked by inhibition of LUBAC activity with the small-molecule HOIL-1-interacting protein (HOIP) inhibitor HOIPIN-8 or by loss of LUBAC catalytic subunit HOIP. Intriguingly, HOIPIN-8, as well as the HOIP inhibitor gliotoxin, and HOIP knockdown effectively prevent TNFα/smac mimetic/zVAD.fmk-induced necroptotic cell death in cells of human origin, without affecting necroptotic RIPK1 and RIPK3 phosphorylation, necrosome formation and oligomerization of phosphorylated MLKL. We demonstrate that HOIPIN-8 treatment inhibits MLKL translocation to intracellular membranes and accumulation in plasma membrane hotspots as well as MLKL exocytosis. We further confirm that HOIPIN-8 treatment suppresses necroptotic cell death in primary human pancreatic organoids (hPOs). Using time-lapse imaging and live/dead staining, we demonstrate loss of organoid structure and hPO cell death induced by smac mimetics and caspase inhibitors, thus providing a novel platform to investigate necroptosis in near physiological settings. Inhibition of LUBAC activity with HOIPIN-8 prevents hPO collapse and extends cell viability. Of note, loss of the M1 Ub-targeting deubiquitinating enzymes (DUBs) OTU DUB with linear linkage specificity (OTULIN) and cylindromatosis (CYLD) in human cell lines does not affect necroptosis induction and HOIPIN-8-mediated rescue of necroptosis. Intriguingly, inhibition of LUBAC activity with HOIPIN-8 does not block necroptotic cell death in murine cell lines.
Using massive analyses of cDNA ends (MACE)-seq-based global transcriptome analysis we confirm that necroptosis induces a pro-inflammatory cytokine profile which is dependent on LUBAC function and necroptotic signaling. Loss of LUBAC activity prevents the MLKL-dependent production and release of pro-inflammatory cytokines and chemokines.
Finally, we identify Flotillin-1 and -2 (FLOT1/2) as putative targets of necroptosis-induced M1 poly-Ub. Ubiquitin-binding in ABIN and NEMO (UBAN)-based pulldowns of M1 poly-ubiquitinated proteins revealed enrichment of FLOTs after necroptosis induction which is dependent on LUBAC activity and can be blocked with necroptosis inhibitors Nec-1s, GSK’872 and NSA, targeting RIPK1, RIPK3 and MLKL, respectively. Of note, loss of FLOT1/2 potentiates necroptosis suppression induced by LUBAC inhibition with HOIPIN-8.
Together, these findings identify LUBAC-mediated M1 poly-Ub as an important mediator of necroptosis and identify FLOTs as novel putative targets of LUBAC-mediated M1 poly-Ub during necroptosis. In addition, by modeling necroptosis in primary human organoids, we further expand the spectrum of experimental models to study necroptosis in human cellular settings.
Nukleäre Rezeptoren (NRs) sind ligandengesteuerte Transkriptionsfaktoren, die sich aus einer Superfamilie von 48 humanen Mitgliedern zusammensetzt. Seit vielen Jahrzehnten stellen sie ein attraktives Forschungsgebiet für die Arzneistoffentwicklung dar, da sie eine bedeutende Rolle in zahlreichen Prozessen unseres Körpers spielen. Das Ziel dieser Forschungsarbeit bestand darin, neue innovative Liganden für den Peroxisomen-Proliferator-aktivierter-Rezeptor γ (PPARγ) sowie die Waisenrezeptoren Nervenwachtumsfaktor induzierter Klon B (Nur77) und Neuronen-abgeleiteter Waisenrezeptor (NOR-1) zu identifizieren.
Bei den Rezeptoren Nur77 und NOR-1 handelt es sich um noch unzureichend erforschte NRs der NR4A-Familie. Es fehlt insbesondere an Modulatoren dieser Rezeptoren als Werkzeuge, um ihr zum Teil noch unentdecktes Potential zu erforschen. Um diese Lücke zu schließen, wurde ein in vitro Screening durchgeführt und eine Arzneistoff-Fragment-Bibliothek mit 480 Fragmenten, die aus bekannten strukturellen Motiven zugelassener Arzneimittel stammen, auf ihre modulatorische Aktivität an Nur77 und NOR-1 gescreent. Durch das Screening und weitere Testungen konnten jeweils für Nur77 und für NOR-1 drei Verbindungen als Liganden identifiziert werden. Bei der weiteren Charakterisierung stellte sich insbesondere 41 als besonders vielversprechenden Ausgangspunkt für die Entwicklung von Liganden für Nur77 und NOR-1 heraus, der ein besseres Verständnis für die invers agonistische Aktivität lieferte und die Möglichkeit für eine agonistische Modulation aufzeigte. Zudem konnte durch ein weiteres Screening mit Computer-gestützten Verfahren auf Nur77 der Chemotyp von 41 noch weiter optimiert werden und führte zur Identifizierung von Verbindung 68 (EC50 = 2 ± 1 μM). Diese zeichnete sich durch eine hohe Potenz aus, die zu einer beachtenswerten Aktivierung von Nur77 (169 ± 18% maximale Aktivierung) führte. Die Untersuchung der strukturellen Erweiterung von 43 (IC50 = 47 ± 8 μM) führte zur Verbindung 75, die eine 3,5-fache Steigerung des inversen Agonismus auf NOR-1 zeigte. Die Erkenntnisse dieser Entdeckung ermöglichte den Rückschluss, dass das Einführen von voluminösen Resten, wie Brom oder Phenyl eine invers agonistische Potenz im unteren mikromolaren Bereich bewirkte. Die Identifizierung der Verbindungen 41 und 68 für Nur77 sowie 43 und 76 für NOR-1 könnten dazu beitragen, ein tieferes Verständnis der molekularen Mechanismen hinter der Aktivierung von Nur77 und NOR-1 zu erlangen und einen vielversprechenden chemischen Ausgangspunkt für die Entwicklung von noch wirksameren und selektiveren Liganden bieten.
Im anderen Teil dieser Forschungsarbeit stand die Synthese eines selektiven allosterischen PPARγ-Liganden im Fokus, um mit diesem die allosterische Modulation von PPARγ zu charakterisieren. Den Ursprung der Idee lieferte Garcinolsäure, dass in der Lage ist, PPARγ orthosterisch und allosterisch zu binden. Aufgrund der komplexen biologischen Effekte und der geringen synthetischen Zugänglichkeit konnte 37 nicht als Ausgangspunkt für dieses Vorhaben dienen. Auf der Suche nach einer geeigneten Ausgangsverbindung wurde durch ein in vitro Screening mit einer hauseigenen Sammlung von synthetischen PPARγ-Modulatoren, bei dem die orthosterische Bindungsstelle von PPARγ durch den irreversiblen Antagonisten GW9662 blockiert wurde, Verbindung 39 identifiziert. Diese ist wie 37 in der Lage PPARγ ortho- und allosterisch zu binden, weist aber eine bessere synthetische Zugänglichkeit auf. Die Co-Kristallisation von 39 mit der PPARγ-Ligandenbindungsdomäne zeigte, dass die orthosterische Bindungstasche (BT) keinen Platz für eine Verlängerung des Moleküls bietet, die allosterische BT ist dagegen Lösungsmittel exponiert, wodurch eine Verlängerung möglich schien. Daraufhin wurde die Hypothese aufgestellt, dass eine Verlängerung von 39 eine orthosterische Bindung verhinderte und dadurch eine selektive allosterische Bindung ermöglichen könnte. Aus diesem Grund wurde eine modifizierte Struktur von 39 verwendet, um eine einfache Einbringung eines Linkers in das Molekül zu ermöglichen. Durch verschiedenste Modifikationen und Anpassungen wurde 104 als potenzieller selektiver allosterischer Ligand synthetisiert. Die Testung von 104 im Reportergenassay zeigte eine schwache Aktivierung von PPARγ allein, jedoch offenbarte sich bei der Kombination mit dem orthosterischen Agonisten Pioglitazon eine dosisabhängige Steigerung der Aktivität von PPARγ. Diese Ergebnisse deuteten darauf hin, dass trotz der Bindung von 104 eine Bindung von 33 in die orthosterische BT immer noch möglich war. Diese Annahme konnte anschließend auch durch zellfreie Experimente (Isotherme Titrationskalorimetrie, MS-basierte-PPARγ-Ligandenbindungs-Assay) bestätigt werden. Der eindeutige Beweis für die selektive allosterische Bindung von 104 lieferte die Co-Kristallisation von 104 mit der PPARγ-LBD. Zusätzlich offenbarte sich, durch den strukturellen Vergleich der Bindungsmodi von anderen PPARγ-Liganden, der außergewöhnliche Bindungsmodus von 104, da 104 im Vergleich zu anderen Liganden selektiv die allosterische BT, ohne Überlappung in die orthosterische BT, besetzte. Weitere Untersuchungen, wie der Einfluss von 104 auf die Rekrutierung von Co-Regulatoren, die Differenzierung von adipozytären Stammzellen und die Genexpression zeigten eine bisher einmalige Modulation von PPARγ, die auf die selektive allosterische Modulation zurückzuführen war. Mit 104 wurde ein innovatives und vielfältig einsetzbares Werkzeug zur Erforschung der allosterischen Modulation von PPARγ entdeckt, dessen Geschichte an diesem Punkt noch nicht zu Ende ist.
The absolute configurations of the diastereomeric 10-hydroxyaloins, which may be regarded as parent structures for other naturally occurring oxanthrone-C-glucosyls, have been established as 10R, 16 R (A) and 10 S, 16 R (B) by an X-ray structure analysis of the A-octaacetyl derivative (C 16 is the anomeric glucosyl carbon atom). The determination was confirmed by CD spectroscopic comparison with the structural analogues aloins A and B, which should prove useful for making future configurational assignments within this class of compounds. A conformational analysis by the use of a molecular modeling method based on force-field calculations reveals the presence of an extra- and an intra-form, the extra-form of which is energetically preferred.
In the course of systematic investigations on sila-substituted parasympatholytics the diphenyl(2-aminoethoxymethyl)silanols 3b and 4b (and its carbon analogue 4a) were synthesized and characterized by their physical and chemical properties. In the solid state 4a and 4b form strong O-H---N hydrogen bonds, which are intramolecular (4a) and intermolecular (4b), respectively. 4a and 4b were found to be weak antimuscarinic agents (4b >4a) and strong papaverine-like spasmolytics (4a ≈4b).
In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).