Eldorado Community:
http://hdl.handle.net/2003/25888
2024-03-29T09:55:10ZCovalent allosteric inhibitors of Akt generated using a click fragment approach
http://hdl.handle.net/2003/42375
Title: Covalent allosteric inhibitors of Akt generated using a click fragment approach
Authors: Westhuizen, Leandi van der; Weisner, Jörn; Taher, Abu; Landel, Ina; Quambusch, Lena; Lindemann, Marius; Uhlenbrock, Niklas; Müller, Matthias P.; Green, Ivan R.; Pelly, Stephen C.; Rauh, Daniel; Otterlo, Willem A. L. van
Abstract: Akt is a protein kinase that has been implicated in the progression of cancerous tumours. A number of covalent allosteric Akt inhibitors are known, and based on these scaffolds, a small library of novel potential covalent allosteric imidazopyridine-based inhibitors was designed. The envisaged compounds were synthesised, with click chemistry enabling a modular approach to a number of the target compounds. The binding modes, potencies and antiproliferative activities of these synthesised compounds were explored, thereby furthering the structure activity relationship knowledge of this class of Akt inhibitors. Three novel covalent inhibitors were identified, exhibiting moderate activity against Akt1 and various cancer cell lines, potentially paving the way for future covalent allosteric inhibitors with improved properties.2022-02-16T00:00:00ZTargeting RNA-protein interactions with peptides and small molecule inhibitors
http://hdl.handle.net/2003/42315
Title: Targeting RNA-protein interactions with peptides and small molecule inhibitors
Authors: Chang, Jen-Yao
Abstract: WD repeat domain 5 (WDR5) is a scaffold protein involved in protein-protein or RNA-protein complexes, and most of these complexes play an important role in various epigenetic modulation processes. In particular, some of the long non-coding RNAs (lncRNAs) rely on the formation of the lncRNA-WDR5 complex to exert their epigenetic modulation, such as the upregulation of the lncRNA itself. If an oncogenic lncRNA relies on this pathway to maintain its expression level, it is possible to inhibit its positive feedback loop and thus reduce the expression of the oncogenic lncRNA. Therefore, this thesis focuses on investigating the potential of targeting lncRNA-WDR5 interactions, followed by evaluating the downregulation of lncRNA in cellulo.
Targeting lncRNA-WDR5 interactions could be achieved by designing an inhibitor that targets the same binding pocket on WDR5. Several lncRNA are reported to recognize WDR5-binding motif (WBM) site, as a result, a study of structure-activity relationship of peptide-based inhibitors derivatize from protein-WDR5 interactions to target WBM site are performed. Further optimizations are performed by tailor-design macrocycle structure to facilitate peptides adopt the binding conformation and indeed the binding affinity is improved. The ability of macrocycle to disrupt lncRNA-protein interaction is verified by competitive in vitro RNA immunoprecipitation (iv-RIP).
Cellular experiments are performed to determine whether targeting lncRNA-WDR5 interactions leads to downregulation of the lncRNA itself. Several strategies show their ability to enhance cell uptake of macrocycles. Finally, two molecules show that targeting lncRNA-WDR5 complexes can lead to reduction of the lncRNA itself and that different lncRNA have different sensitivity to the treatment.2023-01-01T00:00:00ZA bacterial surface display platform for the discovery of cytosine modification readers from cDNA libraries
http://hdl.handle.net/2003/42314
Title: A bacterial surface display platform for the discovery of cytosine modification readers from cDNA libraries
Authors: Schiller, Damian
Abstract: 5-methylcytosine (5mC) occurs in palindromic cytosine guanine dyads (CpGs) of mammalian genomes and is a key element of epigenetic transcription regulation. Central to these regulatory functions is the ability of cytosine modifications to modulate the interaction of chromatin proteins with DNA. In this context, reader proteins are known to selectively recognize the cytosine modification. Interactome profiling studies based on pulldowns of nuclear extracts in combination with mass spectrometry-based (MS) proteomics have been the main approach to discovering readers of 5mC. However, this approach may miss important (anti-)readers due to the competition of proteins for the probe and low expression levels. Moreover, direct readers cannot be distinguished from indirect binders of protein complexes.
In the scope of this thesis, a bacterial surface display system for discovering novel reader candidates from human cDNA was established. In detail, our approach offers screenings of cDNA-encoded protein libraries independent of their endogenous expression levels and without competition with other proteins. It further allows for rapid iterative FACS selections with defined, fluorescently labeled on- and off-target DNA probes. Furthermore, the system benefits from the fast protein expression machinery and growth of bacterial cells. We created surface display libraries of full-length and fragmented coding sequences (CDS) from human cDNA and demonstrated the compatibility of the Intimin surface display platform with displaying human proteins and protein fragments. Moving on, we proved the functionality of our selection system by enriching known 5mC reader candidates from a display library of human thyroid cDNA. In addition, we were able to identify novel reader candidates. Therefore, this study offers a promising tool to complement existing efforts in discovering 5mC reader candidates. Furthermore, the tool bears the potential to be extended to the oxidized derivates of 5mC and their combinations in CpGs, which have not been investigated so far.2023-01-01T00:00:00ZDesign, synthesis and biological evaluation of tool compounds for the cellular investigation of deubiquitinases
http://hdl.handle.net/2003/42255
Title: Design, synthesis and biological evaluation of tool compounds for the cellular investigation of deubiquitinases
Authors: Schmidt, Mirko
Abstract: The ubiquitin proteasomal system, is an important mediator of protein homeostasis through ubiquitination of substrates, but also coordinates various other functions throughout the cell. Deubiquitinases (DUBs) are essential regulators within this system. They are a specialized class of proteases that can cleave ubiquitin from its substrates and their dysregulation is involved in the development of various diseases. However, the understanding regarding their substrates, cellular localization, their involvement in cellular signaling and structural features remains limited. Despite the enormous therapeutical potential only a few DUB inhibitors are known to date. Therefore, novel tool compounds are of urgent need to enhance the understanding of DUBs in a cellular setting.
This thesis describes the discovery, chemical synthesis and biological evaluation of such tool compounds for investigating DUBs in a living environment. To accomplish this, literature-known compounds were resynthesized to introduce alkyne tags to utilize their use as activity-based probes (ABPs) in cell-based systems. An intensive cellular characterization of a dedicated small molecule ABP library targeting DUBs resulted in the discovery of the chemogenomic pair of probes GK13S and GK16S, usable for the selective investigation of the DUB UCHL1 in a living environment. The probes were used to unravel potential substrates and a cellular phenotype after the inhibition of UCHL1. Furthermore, this thesis describes the synthesis and cellular evaluation of PROTAC molecules to study DUBs beyond inhibition.
Altogether, this thesis should foster the understanding of DUBs and serve as a blueprint for the synthesis of further probe molecules to study these enzymes.2023-01-01T00:00:00ZA cell fusion interaction network during the mating of Saccharomyces cerevisiae
http://hdl.handle.net/2003/42197
Title: A cell fusion interaction network during the mating of Saccharomyces cerevisiae
Authors: Hagemeier, Angela
Abstract: Cell-cell fusion is essential for sexual reproduction and occurs when the lipid membranes of two
distinct cells merge into one continuous bilayer. While in recent years some general aspects have
been uncovered, the underlying molecular mechanism remains poorly understood. Mating of
haploid Saccharomyces cerevisiae cells of the opposite sex provides an ideal model system to
study plasma membrane (PM) fusion in eukaryotic organisms. In this work, a multicolor flow
cytometry assay based on fluorescent complementation (BiFC) of split-GFP was adapted to screen
a customized yeast knockout library (YKO) for fusion defects. In total, 28 mutants were identified
that exhibited fusion levels at least as defective as .prm1, a known regulator of this step. Like
.prm1, the majority displayed a bilateral fusion defect.
The remaining part of the work focused on an in-depth analysis of select gene of interest (GOI)
mutants. Investigations of synergistic relationships in trans revealed an interaction network
operating during PM fusion involving at least four independent yet partially overlapping fusion
pathways. Previously two pathways with ERG6 and PRM1 have been reported. VMA2, a gene
encoding a subunit of the vacuolar membrane ATPase (V-ATPase), was revealed in this work to
operate on a third pathway. The findings show that the V-ATPase i) promotes cell fusion indirectly
by acidifying endomembrane organelles, ii) facilitates both cell wall (CW) remodeling and PM
fusion stages approximately equally, and iii) synergistically interacts with 12 other genes identified
in this study. CAX4 was identified to operate on the fourth pathway and the only novel gene found
to synergize with PRM1. Further investigations revealed that Prm1p was less abundant in a .cax4
sensitized background, while its localization is not affected.
Finally, this work discovered that several subunits of the RNA polymerase II mediator complex are
involved in promoting early and late stages of yeast mating. The deletion of subunits leads to
varying degrees of defects in cell pairing and pheromone secretion as well as CW remodeling and
PM fusion. Together, these findings suggest that the mediator complex acts as a master regulator
of cell fusion perhaps by synchronizing the expression of mating genes needed at crucial time
points starting from the digestion of the CW up to the merging of the PMs.2023-01-01T00:00:00ZTranscriptomics-based developmental toxicity and cardiotoxicity assessment using induced pluripotent stem cells
http://hdl.handle.net/2003/42196
Title: Transcriptomics-based developmental toxicity and cardiotoxicity assessment using induced pluripotent stem cells
Authors: Cherianidou, Anna
Abstract: This PhD study focuses on developing an innovative in vitro test system using human-induced pluripotent stem cells (hiPSCs) to assess developmental toxicity. Traditional approaches rely on animal studies, which can be complex, costly, and less applicable to humans. The study introduces the UKK2 protocol combined with hiPSCs and transcriptomics to evaluate both developmental teratogenicity and cardiotoxicity. The research involves differentiating hiPSCs into various germ layers and cardiomyocytes in the presence of substances known to have embryotoxic effects. Gene expression analyses identify gene signatures associated with early embryotoxicity and cardiotoxicity. The study's first phase assesses the effectiveness of the UKK2 test system with 23 teratogens and 16 non-teratogens at varying concentrations. A classifier with high accuracy (90% to 92%) is established for predicting teratogens using the UKK2 test system. The research extends the UKK2 protocol to evaluate developmental cardiotoxicity (UKK-CTT). A gene signature predicts the inhibitory potential of teratogens and non-teratogens in cardiomyogenesis. The "Developmental Cardiotoxicity Index" (CDI31g) effectively distinguishes compounds affecting hiPSC differentiation into functional cardiomyocytes. This study anticipates a promising future for human-based in vitro models in developmental toxicity screening, improving drug testing and teratogenicity prediction. These advancements provide valuable insights into drug-induced developmental toxicity during cardiac organogenesis, enhancing our understanding of critical embryonic development phases and drug safety during vulnerable periods.; Diese Doktorarbeit konzentriert sich auf die Entwicklung eines innovativen In-vitro-Testsystems unter Verwendung von menschlichen induzierten pluripotenten Stammzellen (hiPSCs) zur Bewertung der Entwicklungstoxizität. Herkömmliche Ansätze beruhten auf Tierversuchen, die oft komplex, kostspielig und aufgrund von Speziesunterschieden nicht direkt auf den Menschen übertragbar sind. Das "Universitätsklinikum Köln (UKK)2"-Testsystem wurde entwickelt, um sowohl Entwicklungsteratogenität als auch Kardiotoxizität zu bewerten. Es nutzte hiPSCs, Transkriptom-Analysen und ausgewählte Substanzen in in vivo-ähnlichen Konzentrationen. Die Differenzierung von hiPSCs in verschiedene Keimblätter und Kardiomyozyten wurde in Gegenwart und Abwesenheit von Substanzen mit embryotoxischer Wirkung untersucht. Genexpressionsanalysen identifizierten charakteristische Gene-signaturen für Embryotoxizität und Kardiotoxizität. In der ersten Phase wurden 23 Teratogene und 16 Nicht-Teratogene in maximaler Plasmakonzentration (Cmax) und 20-facher Cmax-Konzentration getestet. Ein prädiktiver Klassifikator mit einer Genauigkeit von 90% bis 92% wurde entwickelt, um Teratogene vorherzusagen. Das UKK2-Testsystem wurde dann zum UKK-Kardiotoxizitätstest (UKK-CTT) erweitert, um Substanzen zu testen, die die Kardiomyogenese hemmen. Eine Gensignatur wurde identifiziert, um Teratogene von Nicht-Teratogenen zu unterscheiden. Der "Developmental Cardiotoxicity Index" (CDI31g) wurde erstellt, um Substanzen zu unterscheiden, die die Kardiomyogenese beeinflussen. Die Studie zeigt vielversprechende Möglichkeiten für hiPSC-basierte In-vitro-Modelle zur Identifizierung von Embryotoxinen in der präklinischen Phase der Arzneimittelentwicklung. Die Entwicklung und Anwendung des UKK2-Testsystems und des UKK-CTT haben wertvolle Erkenntnisse über Entwicklungstoxizität und Kardiotoxizität geliefert und bilden eine Grundlage für die sichere Arzneimittelentwicklung in der präklinischen Phase.2023-01-01T00:00:00ZTargeting of RNA-binding proteins with macrocyclic peptides
http://hdl.handle.net/2003/42190
Title: Targeting of RNA-binding proteins with macrocyclic peptides
Authors: Schmeing, Stefan
Abstract: Die Arzneimittelforschung konzentrierte sich lange Zeit auf die Entwicklung von Modulatoren für Enzyme, da viele Enzyme zielgerichtete, kleine Taschen und katalytische Stellen aufweisen, für die kleine Moleküle auf rationale Weise entworfen oder gescreent werden konnten. Auch Protein-Protein-Wechselwirkungen, für die verschiedene chemische Räume erforscht werden mussten, wurden erfolgreich ins Visier genommen. In den letzten Jahren wurde das Interesse an vielfältigeren makromolekularen Komplexen wie Nukleinsäure Protein-Interaktionen geweckt. Bei dieser Arbeit liegt der Schwerpunkt auf der Entdeckung von Modulatoren von Protein-RNA Interaktionen. Das Projekt konzentrierte sich auf den Spleißfaktor polypyrimidine tract binding protein 1 (PTBP1), der bei Überexpression
verschiedene Krankheiten wie Krebs, Herzerkrankungen, Diabetes und den Abbau von Nervenzellen verursacht. Dieses Protein besteht aus vier RNA bindenden Domänen, die unterschiedliche Konsensussequenzen mit hoher Ähnlichkeit binden und durch die Tertiärstruktur des Proteins und der RNA eine komplexe Bindung an die Ziel-RNAs ermöglichen. Zunächst wurde versucht, einen auf E. coli basierenden zellulären Assay zur Quantifizierung der Interaktion von PTBP1 mit seinen Ziel-RNAs zu entwickeln. Dieser Assay sollte verwendet werden, um eine Bibliothek genetisch kodierter Peptidmakrozyklen auf Inhibitoren dieser Interaktion zu untersuchen. Später wurde die Funktion einer kürzlich berichteten transienten α-Helix zwischen den N-terminalen Domänen von PTBP1 untersucht und genutzt, um stabilisierte Peptide zu entwickeln, die mit der nativen, in das Protein eingebetteten Helix in vitro konkurrieren. Es konnte gezeigt werden, dass die Makrozyklen die Kooperativität zwischen RRM1 und RRM2 für eine RNA mit zwei Bindungsstellen hemmen. Die Peptide weisen einen einstelligen mikromolaren KI in den durchgeführten Experimenten auf. Diese Moleküle erwiesen sich als mäßig zellpermeabel, lysatstabil und modulierten das Spleißen mit geringen Auswirkungen. Darüber hinaus wurde der Bindungsmodus eines Peptids durch Ko-Kristallisation mit RRM1 validiert.
In einem weiteren Projekt wurden makrozyklische Peptid-Inhibitoren für ein nicht konventionelles RNA-Bindungsprotein WDR5 strukturell untersucht, um ihre Wirkungsweise zu validieren.; Drug discovery traditionally focused on the development of modulators for enzymes for a long time because many enzymes provide targetable small pockets and catalytic sites for which small molecules could be rationally designed or screened. Also, protein-protein interactions, for which different chemical spaces needed to be explored, were targeted successfully. In the recent years the interest in more diverse macromolecular complexes like nucleic acid-protein interactions awaked interest. For this work, the focus is on the discovery of modulators of protein-RNA interactions. The project focused on the splicing factor polypyrimidine tract binding protein 1 (PTBP1) which drives several diseases including cancer, cardiac diseases, diabetes and neuronal degradation when overexpressed. This protein consists of four RNA recognition motifs which bind to distinct consensus sequences with high similarities, which orchestrate a complex binding mode to target RNAs through the tertiary structure of the protein and RNA. First, the development of an E. coli based cellular assay for the quantification of the interaction of PTBP1 with its target RNAs was attempted. This assay was planned to be used to screen a library of genetically encoded peptide macrocycles as inhibitors of this interaction. Later, the function of a recently reported transient α-helix between the N-terminal domains of PTBP1 was investigated and utilized to design stapled peptides to compete with the native helix in vitro. Here, it was shown that the macrocycles inhibit the cooperativity between RRM1 and RRM2 which compete with a single RNA molecule with two binding sites with a single digit micromolar KI. These molecules were validated to have a sufficient cell-permeability, lysate stability, and modulated splicing with low effects. Further on, the binding mode of one peptide was validated through co-crystallization with RRM1 lacking the native helix.
In another project, macrocyclic peptide inhibitors for a non-conventional RNA binding protein WDR5 was structurally studied to validate their mode of action.2023-01-01T00:00:00ZA primer on building life-like systems
http://hdl.handle.net/2003/42174
Title: A primer on building life-like systems
Authors: Vibhute, Mahesh A.; Mutschler, Hannes
Abstract: The quest to understand life and recreate it in vitro has been undertaken through many different routes. These different approaches for experimental investigation of life aim to piece together the puzzle either by tracing life's origin or by synthesizing life-like systems from non-living components. Unlike efforts to define life, these experimental inquiries aim to recapture specific features of living cells, such as reproduction, self-organization or metabolic functions that operate far from thermodynamic equilibrium. As such, these efforts have generated significant insights that shed light on crucial aspects of biological functions. For observers outside these specific research fields, it sometimes remains puzzling what properties an artificial system would need to have in order to be recognized as most similar to life. In this Perspective, we discuss properties whose realization would, in our view, allow the best possible experimental emulation of a minimal form of biological life.2022-12-08T00:00:00ZIdentification of small molecule inhibitors of the kynurenine pathway
http://hdl.handle.net/2003/42149
Title: Identification of small molecule inhibitors of the kynurenine pathway
Authors: Dötsch, Lara Jil
Abstract: The immune system functions as an effective barrier against tumor development. However, malignant cells can avoid elimination by the immune system by acquiring certain characteristics that alter the immune system in the tumor microenvironment (TME). For instance, cancer cells can actively induce immune tolerance by expression of the immuno-suppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 expression results in degradation of the essential amino acid tryptophan (Trp) and production of kynurenines (Kyn) via the Kyn pathway. Targeting the Kyn pathway has emerged to be an attractive target in immuno-oncology.
In this thesis, two compound classes were discovered through a cell-based screening that reduce Kyn levels in cancer cells upon stimulation with the cytokine interferon-γ (IFN-γ) by two distinct mechanisms. The indole-tetrahydropyridine pseudo-natural products (PNPs) called apoxidoles inhibit IDO1 directly by binding and stabilizing apo-IDO1 in vitro and in cells. The displacement of the IDO1 cofactor heme catalytically inactivates the enzyme and thereby decreases Kyn levels. The second small molecule class of N-substituted indoles called epoxykynins represents an alternative approach to modulate the Kyn pathway by not directly targeting IDO1. Instead, epoxykynin inhibits the catalytic activity of the C-terminal fatty acid hydrolase domain of the soluble epoxide hydrolase (sEH-H). As part of the arachidonic acid (AA) cascade, the highly active enzyme sEH-H plays an important role in the hydrolysis of CYP epoxygenase-derived fatty acid epoxides. Thereby, it contributes to the regulation of bioavailable epoxides and controls a variety of biological processes, such as inflammation, vasodilation, angiogenesis, neuropsychiatries and pain. Thereby, it contributes to the regulation of bioavailable epoxides and controls a variety of biological processes, such as inflammation, vasodilation, angiogenesis, neuropsychiatries and pain. The results obtained throughout this thesis uncover a cross-talk between sEH-H and the Kyn pathway.
The discovery of new Kyn pathway inhibitors is in high demand, since the most advanced holo-IDO1 inhibitor epacadostat has recently failed in clinical trials. Additionally, the identification of epoxykynins has deepened the understanding of the functional link between the Kyn pathway and the AA metabolism. These findings might enable novel strategies to design immunotherapies and enhance the host’s immune system to overcome cancer-induced immune tolerance.2023-01-01T00:00:00ZDiscovery of small-molecule modulators of the RNA-binding proteins LIN28 and RNase L
http://hdl.handle.net/2003/42135
Title: Discovery of small-molecule modulators of the RNA-binding proteins LIN28 and RNase L
Authors: Borgelt, Lydia
Abstract: RNA-binding proteins (RBPs) regulate all aspects of RNA biology and metabolism and are emerging targets for the development of molecular probe compounds and therapeutics for various diseases. While oligonucleotide-based and peptide-based strategies have been applied to modulate RNAs and protein–RNA interactions, efficient RBP-targeting strategies utilizing small molecules, which bear inherent merits in comparison with other chemotypes, remain obscure. In this thesis, I studied small-molecule-based strategies to target three different RBPs, the oncogenic miRNA-binding protein LIN28, the antiviral ssRNA-cleaving protein RNase L and the associated dsRNA-binding protein OAS.
LIN28, which negatively regulates let-7 miRNAs that downregulate the translation of numerous oncogenic proteins, is overexpressed in many human cancers and is a driver of tumor progression and metastasis. Thus, LIN28 inhibition via small molecules is a promising strategy for the development of cancer therapeutics. Reported LIN28 inhibitors suffer from poor inhibitory potency, insufficient characterization of mechanism of action, limited structure-activity relationship, and poor cellular activity. This work employed a screening-based and a scaffold-based approach for the identification of LIN28 inhibitors with new scaffolds and improved potency. In the former approach, trisubstituted pyrrolinones were identified as LIN28 inhibitors via a fluorescence polarization assay-based screening. The most active pyrrolinone 41 increased the expression levels of mature let-7 in LIN28-expressing cells. A following structure-activity relationship study revealed biphenyl compounds, such as 85, that showed a more potent effect in inducing let-7 maturation. In the latter approach, a spirocyclization strategy was applied based on the chromenopyrazole scaffold of reported LIN28 inhibitors to increase the LIN28-inhibitory potency induced by the rigidity of the spirocyclic scaffold. The identified and well-characterized inhibitors are worthy starting points for the development of anticancer drugs and LIN28-targeting chemical probes.
The dsRNA-cleaving protein RNase L and the ssRNA-binding protein OAS are key enzymes in the human antiviral innate immune response. OAS detects foreign dsRNA upon viral infection, leading to OAS activation and production of the second messenger 2’-5’A. The binding of 2’-5’A to RNase L leads to RNase L dimerization and activation. The RNase L dimer then cleaves ssRNAs resulting in a global translational arrest and a cellular antiviral state. Small-molecule activators of OAS and RNase L are thus promising candidates for the development of broad-spectrum antiviral therapeutics. Furthermore, RNase L activators are useful components to build bifunctional RNase L recruiters to achieve proximity-induced targeted degradation of RNAs. In this work, robust assays for the screening and validation of OAS and RNase L activators were developed and used for the discovery of small-molecule modulators. While the identification of OAS and RNase L activators proved to be challenging, potential RNase L binders were identified. Additionally, a rational design approach led to the discovery of thiophenones as RNase L inhibitors that showed more potent inhibitory potency than reported RNase L inhibitors that target the nucleotide-binding pocket of RNase L
Collectively, the results of this thesis demonstrate the feasibility targeting RBPs using a variety of small-molecule-based strategies. Screening- and scaffold-based approaches enabled the identification of modulators with new scaffolds and improved potency in targeting RBPs. The established assays, the identified compounds, and the optimization strategies presented in this work will be useful for the future development of small molecules targeting RBPs.2023-01-01T00:00:00ZStructural investigation of cholesterol homeostasis and bacterial toxins
http://hdl.handle.net/2003/42108
Title: Structural investigation of cholesterol homeostasis and bacterial toxins
Authors: Günther, Patrick
Abstract: Membrane proteins regulate a variety of processes that are critical for living organisms. They participate in cell-cell communication, catalyze reactions in or at the membrane, are involved in transmitting signals from the environment into the cell, and can transport molecules across membranes. Approximately 60% of all clinically approved drugs target membrane proteins, underscoring their importance. In order to understand the function of membrane proteins and to design more targeted drugs, determining their precise three-dimensional structures is required. In this PhD project, I aimed to structurally characterize two membrane protein complexes involved in the regulation of cholesterol homeostasis – the Scap-Insig and HMGCR-UBIAD1 complexes – and the type VI secretion system (T6SS) effector RhsA. My PhD work showcases that biochemical studies combined with structural determination by cryo-EM provides valuable insights into molecular processes that occur in or at the membrane and is of utmost pharmacological interest.2023-01-01T00:00:00ZProbing RNA catalysis in protocellular settings relevant to the origin of life
http://hdl.handle.net/2003/42093
Title: Probing RNA catalysis in protocellular settings relevant to the origin of life
Authors: Salibi, Elia
Abstract: The emergence of life has long been a topic of high interest. Of the many hypotheses pro-posed for the emergence of early cellular predecessors of our last universal common ances-tor (LUCA), the RNA World hypothesis is a prime candidate. It posits a time before the evolution of modern DNA / protein-based life, where both roles of information storage and processing were played by RNA, while DNA and complex proteins had not yet evolved. With the advent of in vitro evolution, selection experiments have yielded a variety of dif-ferent catalytic RNAs (ribozymes) able to catalyse a range of chemical reactions in a se-quence-dependent manner. Nonetheless, a number of outstanding problems remain, such as the dissemination of genetic information among protocell populations in the absence of complex cell division machinery, genotype-phenotype coupling in RNA-peptide based membrane-less compartments and finally, the general sequence-independent templating abilities of ribozymes. In this work, cycles of freezing and thawing were employed to drive the proliferation of encapsulated RNA replicators among feedstock vesicles, allowing their survival following multiple rounds of dilution via self-amplification, thereby enabling a primitive form of lateral gene transfer among protocell populations and eliminating the need for sophisticated cell division apparatus. In the following section, a ribozyme ligase was re-engineered to perform concatenated ligation of an RNA substrate leading to a drastic increase in length. Ribozyme activity was subsequently characterised in the presence of short lysine homopeptides that allow phase separation and RNA partitioning. Moreover, ribozyme activity was found to modulate the physical properties of coacervates, demon-strating a primitive form of genotype-phenotype linkage in membraneless model protocells. Finally, a group I intron-derived ribozyme ligase that performs templated ligation reaction was employed as a general templated RNA ligase and characterised for the synthesis of a variety of functional RNA sequences. The system was subsequently adopted by collabora-tors as a starting point for the development of environmentally-driven RNA replication systems. Taken together, the data presented here offer insight into the possible mechanisms for persistence and proliferation of prebiotically relevant protocell models in environmental settings as well as an overview of the ‘proto-biosphere’ on early Earth and potential transi-tional stages of chemical and biological evolution leading to the emergence of the first cellular systems as we know them.; Die Entstehung des Lebens ist seit langem ein Thema von großem Interesse. Von den vielen Hypothesen, die für die Entstehung früher zellulärer Vorläufer unseres letzten gemeinsamen Vorfahren vorgeschlagen wurden, ist die RNA-Welt-Hypothese ein Hauptkandidat. Sie geht von einer Zeit vor der Evolution des modernen DNA-/Protein-basierten Lebens aus, in der die RNA sowohl die Rolle der Informationsspeicherung als auch die der Informationsverarbeitung übernahm, während sich die DNA und komplexe Proteine noch nicht entwickelt hatten. Mit dem Aufkommen der In-vitro-Evolution haben Selektionsexperimente eine Vielzahl verschiedener katalytischer RNAs (Ribozyme) hervorgebracht, die in der Lage sind, eine Reihe chemischer Reaktionen in sequenzabhängiger Weise zu katalysieren. Dennoch gibt es noch eine Reihe offener Probleme, wie z. B. die Verbreitung genetischer Informationen zwischen den Populationen von Protozellen in Abwesenheit einer komplexen Zellteilungsmaschinerie, die Genotyp-Phänotyp-Kopplung in membranlosen Kompartimenten auf RNA-Peptidbasis und schließlich die allgemeinen sequenzunabhängigen Template-Fähigkeiten von Ribozymen. In dieser Arbeit wurden Zyklen des Einfrierens und Auftauens eingesetzt, um die Vermehrung eingekapselter RNA-Replikatoren in den Ausgangsvesikeln anzutreiben und ihr Überleben nach mehreren Verdünnungsrunden durch Selbstamplifikation zu ermöglichen, wodurch eine primitive Form des lateralen Gentransfers zwischen Protozellpopulationen ermöglicht wird und die Notwendigkeit eines hochentwickelten Zellteilungsapparats entfällt. Im folgenden Abschnitt wurde eine Ribozym-Ligase so umgestaltet, dass sie eine verkettete Ligation eines RNA-Substrats durchführt, was zu einer drastischen Zunahme der Länge führt. Die Ribozymaktivität wurde anschließend in Gegenwart von kurzen Polylysin-Polymeren charakterisiert, die eine Phasentrennung und RNA-Verteilung ermöglichen. Darüber hinaus wurde festgestellt, dass die Ribozymaktivität die physikalischen Eigenschaften von Koazervaten moduliert, was eine primitive Form der Genotyp-Phänotyp-Verknüpfung in membranlosen Modell-Protozellen demonstriert. Schließlich wurde eine von einem Gruppe-I-Intron abgeleitete Ribozym-Ligase, die eine matrizenabhängige Ligationsreaktion durchführt, als allgemeine RNA-Ligase eingesetzt und für die Synthese verschiedener funktioneller RNA-Sequenzen charakterisiert. Das System wurde anschließend von Kollaborationspartnern als Ausgangspunkt für die Entwicklung von umweltgesteuerten RNA-Replikationssystemen übernommen. Zusammengenommen bieten die hier vorgestellten Daten einen Einblick in mögliche Persistenz- und Vermehrungsstrategien präbiotisch relevanter Protozellmodellen sowie einen Überblick über eine mögliche "Proto-Biosphäre" auf der frühen Erde und potentielle evolutionäre Zwischenstufen, die zur Entstehung der ersten zellulären Systeme geführt haben könnten.2023-01-01T00:00:00ZInvestigation of signal networks that control dynamic cell shape changes
http://hdl.handle.net/2003/42044
Title: Investigation of signal networks that control dynamic cell shape changes
Authors: Nanda, Suchet
Abstract: Cells process information via complex signal networks that include multiple components. Knowledge, about the spatio-temporal organization of these components and their activity state is critical to understand how these signal networks process information. Signal networks involving Rho GTPases play a key role in the spatio-temporal coordination of cytoskeletal dynamics during cell migration. Previous studies that directly investigated the crosstalk between the major Rho GTPases Rho, Rac and Cdc42 revealed a strong increase in Rho activity after Rac1 activation. This Rac1-Rho crosstalk might play a role in mediating the tight spatio-temporal coupling between cell protrusions and retractions that are typically observed during mesenchymal cell migration. To address this question, improved sensors were developed in this thesis to measure the activity state of endogenous small GTPases of the Ras, Rap, Rac, and Rho families in living cells. Using these sensors, Rac activation was observed to be tightly and precisely coupled to local cell protrusions, followed by Rho activation during retraction. In a screen for potential crosstalk mediators, a subset of the Rho activating Lbc-type GEFs, Arhgef11 and Arhgef12, were found to be enriched at the cell periphery during protrusions-retractions cycles. Furthermore, via an optogenetic approach, these Lbc GEFs were observed to recruit to the plasma membrane by active Rac1, suggesting that they indeed might link the cell protrusion signal Rac and the cell retraction signal Rho. Furthermore, depletion of these GEFs via RNA interference impaired cell protrusion-retraction dynamics, which was accompanied with a decrease in migration distance and an increase in migration directionality. These results show that Arhgef11 and Arhgef12 facilitate effective exploratory cell migration by coordinating the cell morphogenic processes of cell protrusion and retraction by coupling the activity of the associated small GTPases Rac and Rho.
Typical activity sensor designs including the improved small GTPase activity sensors described above are limited in the number of readouts that can be combined simultaneously inside a single cell. To extend this number, a new programmable single stranded mRNA-based sensor design was developed. These sensors enable parallel measurements of multiple protein-protein interactions that are dependent on signal network activity states inside a single living cell. As a proof-of-concept, protein kinase A activity sensors were developed, which allowed to distinguish activity dynamics of their different regulatory subunits in parallel inside living cells. Furthermore, RNA scaffold- based activity sensors for small GTPases were developed which enabled monitoring of the activity kinetics of Ras and Rap during pharmacological perturbations. RNA scaffolds, which were functionalized with dominant positive Rac or Rho GTPases enabled a method to evaluate the specificity of effector molecules in parallel inside living cells. These results show that the application of programmable RNA scaffolds can provide critical information about signal network components inside individual, living cells, which cannot be obtained via available standard methods. Such information can be critical to decipher the spatio- temporal organization of complex signal networks inside cells.2023-01-01T00:00:00ZImaging-based analysis of 5-methylcytosine at low-repetitive genomic loci using transcription activator-like effector probes
http://hdl.handle.net/2003/42021
Title: Imaging-based analysis of 5-methylcytosine at low-repetitive genomic loci using transcription activator-like effector probes
Authors: Jung, Anne
Abstract: 5-Methylcytosine (5mC) is the main epigenetic modification of mammalian genomes. It plays significant roles during cell development and differentiation and is involved in the regulation of essential cellular processes such as the control of gene expression. Dysregulation of methylation can lead to aberrant epigenetic patterns associated with a variety of diseases. To analyze cellular 5mC in situ, fluorescently labeled transcription-activator-like effector (TALE) proteins can be used as 5mC-sensitive probes in imaging studies. TALEs are DNA-binding proteins that provide sequence and 5mC selectivity via a domain of modular repeats, each recognizing a specific nucleobase. This enables the design of TALE probes for sequence-specific analysis of 5mC in user-defined target sequences. In imaging studies, 5mC-sensitive and 5mC-insensitive TALE pairs are used in co-stainings to allow the analysis of 5mC independently of changes in target accessibility. However, until now this has been limited to highly repetitive genomic DNA sequences. To extend this approach for the analysis of 5mC in low-repetitive coding gene loci, this work develops a straightforward signal amplification strategy to increase the imaging sensitivity with TALEs. This is achieved by additional immunostaining of the employed TALE probes, enabling the imaging of only 32 theoretical repeat sequences in the low repetitive MUC4 gene locus. In co-staining experiments, this allows the detection of 5mC changes in this locus between cell types with different methylation levels, introduced by DNA methyltransferase knockouts or overexpression. The ability to detect 5mC differences in this small number of target sequences opens up new perspectives for the analysis of 5mC in non-repetitive genomic loci, providing new insights into the regulation of gene expression.2023-01-01T00:00:00ZModification of peptides for improved membrane permeability and side chain diversity
http://hdl.handle.net/2003/42004
Title: Modification of peptides for improved membrane permeability and side chain diversity
Authors: Openy, Oweta Joseph Noah
Abstract: Fortschritte in der Arzneimittelforschung haben zur raschen Entdeckung potenter Hit-Peptide geführt, die bevorzugte Gerüste für die gezielte Beeinflussung von Protein-Protein-Wechselwirkungen (PPI) oder Protein-Nukleinsäure-Wechselwirkungsstellen darstellen, bei denen es sich in der Regel um große, funktionslose hydrophobe Schnittstellen handelt. Hit-Peptide, die typischerweise in den chemischen Raum der "beyond- rule-of-five" (bRo5) fallen und hauptsächlich aus kanonischen Aminosäuren bestehen, haben in der Regel eine schlechte Pharmakokinetik und Membranpermeabilität.
In Kapitel 2 haben wir versucht, die Membranpermeabilität von Peptiden durch den Einbau von (hetero)aromatischen Linkern in das Peptidgerüst zu verbessern. Als Modellpeptid wurde Sanguinamid A (1) ausgewählt, ein membrandurchlässiges zyklisches Peptid marinen Ursprungs mit einer angemessenen berichteten oralen Bioverfügbarkeit (F = 7 ± 4 %), das einen Thiazolring in seinem Rückgrat aufweist. Sein cysteinhaltiges homodetisches Peptidanalogon wurde als Negativkontrolle entwickelt, während das tert-Butylglycin-haltige Analogon, Danamid F (2, F = 51 ± 9%), unsere Positivkontrolle war. Auf eine Verbindungsbibliothek, die aus Pyridin, Pyridin-N-Oxid und 3-Aminobenzoesäure-Linkern bestand, folgte ein Screening der Membranpermeabilität mittels IAM-Chromatographie. Das Pyridin-haltige Analogon 21 wies einen mit Sanguinamid A vergleichbaren IAM-Wert auf, was auf eine gute Membranpermeabilität hindeutet. Darüber hinaus zeigten1 H-NMR-Studien bei variabler Temperatur, dass die Amide im Rückgrat von 21 ein Wasserstoffbrückenbindungs-HB-Netzwerk bilden könnten, das sich von dem von Sanguinamid A unterscheidet, aber dynamisch ist, um Chamäleonität zu ermöglichen.
In Kapitel 3 befassten wir uns mit der späten Funktionalisierung von Peptiden, um unnatürliche Aminosäuren (UAAs) mit Hilfe von Katritzky-Salzen einzuführen. Bei dieser Methode wurden Lysinreste in Katritzky-Salze umgewandelt, gefolgt von photochemischen Giese-Reaktionen unter milden Bedingungen. Die Methode war mit allen kanonischen Aminosäuren und handelsüblichen Harzen und Linkern kompatibel, mit Ausnahme des 2-Chlornitrit-Chlorid-Harzes. Es wurde über einen breiten Substratbereich berichtet, und die Nützlichkeit der Methode wurde durch die selektive Modifizierung eines von zwei Lysinresten des Histon-3-Schwanzpeptids (Reste 1-10), eines von zwei Ornithinresten des zyklischen antibiotischen Peptids Gramicidin S und die Modifizierung des längeren p53-Peptids (15-29) nachgewiesen.2023-01-01T00:00:00ZRibozyme activity modulates the physical properties of RNA–peptide coacervates
http://hdl.handle.net/2003/41990
Title: Ribozyme activity modulates the physical properties of RNA–peptide coacervates
Authors: Le Vay, Kristian Kyle; Salibi, Elia; Ghosh, Basusree; Tang, TY Dora; Mutschler, Hannes
Abstract: Condensed coacervate phases are now understood to be important features of modern cell biology, as well as valuable protocellular models in origin-of-life studies and synthetic biology. In each of these fields, the development of model systems with varied and tuneable material properties is of great importance for replicating properties of life. Here, we develop a ligase ribozyme system capable of concatenating short RNA fragments into long chains. Our results show that the formation of coacervate microdroplets with the ligase ribozyme and poly(L-lysine) enhances ribozyme rate and yield, which in turn increases the length of the anionic polymer component of the system and imparts specific physical properties to the droplets. Droplets containing active ribozyme sequences resist growth, do not wet or spread on unpassivated surfaces, and exhibit reduced transfer of RNA between droplets when compared to controls containing inactive sequences. These altered behaviours, which stem from RNA sequence and catalytic activity, constitute a specific phenotype and potential fitness advantage, opening the door to selection and evolution experiments based on a genotype–phenotype linkage.2023-06-16T00:00:00ZUnderstanding mechanisms of membrane protein Prm1 for membrane fusion during cell-cell fusion of Saccharomyces cerevisiae
http://hdl.handle.net/2003/41914
Title: Understanding mechanisms of membrane protein Prm1 for membrane fusion during cell-cell fusion of Saccharomyces cerevisiae
Authors: Shek, Anson Hao Tian2023-01-01T00:00:00ZTargeting oncogenic KRasG13C with nucleotide-based covalent inhibitors
http://hdl.handle.net/2003/41415
Title: Targeting oncogenic KRasG13C with nucleotide-based covalent inhibitors
Authors: Goebel, Lisa; Kirschner, Tonia; Koska, Sandra; Rai, Amrita; Janning, Petra; Maffini, Stefano; Vatheuer, Helge; Czodrowski, Paul; Goody, Roger S.; Müller, Matthias Philipp; Rauh, Daniel
Abstract: Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer.2023-04-12T00:00:00ZA high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2
http://hdl.handle.net/2003/41311
Title: A high-throughput effector screen identifies a novel small molecule scaffold for inhibition of ten-eleven translocation dioxygenase 2
Authors: Palei, Shubhendu; Weisner, Jörn; Vogt, Melina; Gontla, Rajesh; Buchmuller, Benjamin; Ehrt, Christiane; Grabe, Tobias; Kleinbölting, Silke; Müller, Matthias; Clever, Guido H.; Rauh, Daniel; Summerer, Daniel
Abstract: Ten-eleven translocation dioxygenases (TETs) are the erasers of 5-methylcytosine (mC), the central epigenetic regulator of mammalian DNA. TETs convert mC to three oxidized derivatives with unique physicochemical properties and inherent regulatory potential, and it initializes active demethylation by the base excision repair pathway. Potent small molecule inhibitors would be useful tools to study TET functions by conditional control. To facilitate the discovery of such tools, we here report a high-throughput screening pipeline and its application to screen and validate 31.5k compounds for inhibition of TET2. Using a homogenous fluorescence assay, we discover a novel quinoline-based scaffold that we further validate with an orthogonal semi-high throughput MALDI-MS assay for direct monitoring of substrate turnover. Structure–activity relationship (SAR) studies involving >20 derivatives of this scaffold led to the identification of optimized inhibitors, and together with computational studies suggested a plausible model for its mode of action.2022-09-02T00:00:00ZA protein-based method for he encapsulation of synthetic cytoplasms
http://hdl.handle.net/2003/41292
Title: A protein-based method for he encapsulation of synthetic cytoplasms
Authors: Seidel, Hans2021-01-01T00:00:00ZTheory of biochemical information processing with transients
http://hdl.handle.net/2003/41268
Title: Theory of biochemical information processing with transients
Authors: Yadav, Manish
Abstract: Cells in tissues and organisms operate in dynamic environments, continuously sensing and responding to time-varying chemical signals. In order to accurately interpret the complex information from their environment, biochemical networks in single cells actively process these extracellular signals in real-time. The current concept of biochemical computations places a strong focus on attractor based information processing in cells. Recent studies however have shown that cells generate completely opposite phenotypic responses depending upon frequency of the growth factor, independent of growth factor identity. This breaks down the steady-state description of biochemical information processing. Therefore, we propose to describe biochemical networks embedded in non-stationary environments as non-autonomous systems whose solutions are the dynamic input-dependent trajectories. We show that memory arising through metastable states will enable the system to integrate time-varying signals such that, inputs resulting in different phenotypic responses will be uniquely encoded in phase-space trajectories. The extracellular information of different phenotypes is spread throughout the large signaling networks and represented by characteristically different classes of phase-space trajectories. This encoded information will further be decoded downstream by early response genes (ERG) in real-time, where we show that the feed-forward structure of ERG is sufficient for this task.2022-01-01T00:00:00ZDevelopment and evaluation of small molecule- and bifunctional modulators targeting the ribonuclease L for RNA degradation
http://hdl.handle.net/2003/41257.2
Title: Development and evaluation of small molecule- and bifunctional modulators targeting the ribonuclease L for RNA degradation
Authors: Hwang, Jimin
Abstract: Ribonucleases are one of the critical regulatory enzymes of RNA-involved metabolism in cells and are becoming promising therapeutic targets due to the importance of RNA in many disease mechanisms. In particular, ribonuclease L (RNase L) has attracted great attention as a regulatory enzyme engaged in innate immune responses against viral infections. RNase L plays a vital role in the antiviral response by degrading RNAs, which is suppressed by viruses over evolution. Therefore, activating the RNase L and associated innate immunity by using small molecule-based strategies are one of the promising therapeutic approaches for not only antiviral effect but also RNase L-related anticancer effect. Meanwhile, RNase L-inhibition associates therapeutic potential in treating autoimmune and inflammatory disorders. Although a few small molecule-based RNase L modulators have been reported, the scarcity of potency and selectivity of reported modulators highly requires a thorough optimization and discovery of new scaffolds as well as development of new approaches. In this study, we pursued our efforts in acquiring potent small-molecule modulators of RNase L via two different strategies. The first approach based on small molecule-ligand pursued improved potency of ligands. The second approach was to introduce new chemical modalities to modulate RNase L activity via bifunctional molecules. In the small molecule-ligand approach, we assessed whether scaffold-based design, structure-activity relationship (SAR) study, and rational design could facilitate the generation of a more potent modulator. The scaffold-based design yielded chemically intriguing scaffolds but with no RNase L activity change. The SAR approach yielded compounds which revealed improved binding to RNase L and showed RNase L-mediated cellular downstream effects. The rational design led to a promising fusion scaffold of RNase L modulators, 2-((pyrrol-2-yl)methylene)thiophen-4-one, and compounds which exhibited 30-fold improved inhibitory effect than reported compound in in vitro biochemical evaluation and a potent cellular inhibitory effect. In our second approach, the three bifunctional molecules consist of a homodimerizer, a heterobivalent molecule, and the PROTAC molecule. The homodimerizer resulted in limited improvement of activator potency while heterobivalent molecule did not lead to activity change. Lastly, PROTAC molecules to degrade an RNase L-inhibiting protein have been synthesized. The effect of the PROTAC molecules on the RNase L activation will be evaluated in a future study. We focused on multiple approaches to modulate the challenging but promising target RNase L. This study will contribute to the development of new chemical entities for a potent RNase L modulator and thus for an innate immunity modulator.2023-01-01T00:00:00ZDynamical basis of cellular sensing and responsiveness to spatial-temporal signals
http://hdl.handle.net/2003/41255
Title: Dynamical basis of cellular sensing and responsiveness to spatial-temporal signals
Authors: Nandan, Akhilesh Padmanabhan
Abstract: Under physiological conditions, cells continuously sense and migrate in response to chemoattractant signals that are noisy, conflicting, and changing over time and space. This suggests cells exhibit seemingly opposed characteristics, such as robust maintenance of polarized state longer than the signal duration, while still remaining adaptive to novel signals. However, the dynamical mechanism that enables such sensing capabilities is still unclear. In this thesis, I propose a generic dynamical mechanism based on critical positioning of receptor signaling network in the vicinity of saddle-node of a sub-critical pitchfork bifurcation (SubPB mechanism). The critical organization leads to the emergence of a dynamical "ghost" that gives transient memory in the polarized response, as well as the ability to continuously adapt to changes in signal localization. Using weakly nonlinear analysis, an analytical description of the necessary conditions for the existence of this mechanism in a general receptor network is provided. Comparing to three classes of existing mathematical models for polarization that operate on the principle of stable attractors, I demonstrate that the metastability arising from "ghost" in the SubPB mechanism uniquely enables sensing dynamic spatial-temporal signals in a history-dependent manner. By using a physical model that couples signaling to morphology, I demonstrate how this mechanism enables cells to navigate in changing environments. Using the well characterized Epidermal growth factor receptor (EGFR) sensing network in epithelial cells, I demonstrated that the described transient memory in signaling mimics working memory in neurons, enabling cells to process non-stationary signals.2022-01-01T00:00:00ZDesign, synthesis, and evaluation of small molecules and associated bifunctional conjugates targeting the protein–RNA interaction of LIN28 and let-7
http://hdl.handle.net/2003/41230
Title: Design, synthesis, and evaluation of small molecules and associated bifunctional conjugates targeting the protein–RNA interaction of LIN28 and let-7
Authors: Hommen, Pascal
Abstract: RNAs have evolved as prominent targets to tackle challenging topics in the field of chemical biology and medicinal chemistry. Concomitantly, RNA binding proteins (RBPs) are emerging as a new class of drug targets given the essential regulatory functions of RBPs in deciding cell fates. Despite the increasing understanding of the importance of RBPs, limited chemical tools are available to probe the biological functions of RBPs, which raises the need for the development of effective and selective tool compounds targeting RBPs. The LIN28–let-7 interaction is one of the most well-investigated protein–RNA interactions to date. Due to its association with a poor cancer prognosis, LIN28 is a potential new anticancer target. Among the current collection of reported LIN28 inhibitors, potent molecules with clear mechanisms of inhibition are lacking. Additionally, most reported LIN28 inhibitors were identified via high throughput screening of different formats against molecular libraries that were not tailored to target RBPs. Therefore, there is a need to diversify the discovery approaches for RBP-targeting molecules. In this context, small-molecule-based approaches were adopted to engage the challenging topic of targeting the miRNA-binding protein LIN28. Furthermore, we designed an intriguing bifunctional molecule in which an affinity-enhancing moiety was linked to a known LIN28 small-molecule inhibitor to build bifunctional molecules to improve efficacy. Inspired by a rationale, first established in targeting protein–protein interactions, hotspot amino acids were identified through an analysis of LIN28–let-7 crystal structure and a virtual alanine scan to design the corresponding affinity-enhancing moieties consisting of peptides. Conjugation between the designed peptides and the known LIN28 inhibitors led to enhanced binding affinity. In other approaches, we investigated compounds of three chemical scaffolds intending to study the structural features required for LIN28 inhibition, as well as identifying suitable small molecules to be used in the bifunctional molecule approach. After the initial investigation, chromeno[4,3-c]pyrazoles were used as the small molecule components to be conjugated to a peptide moiety through CuAAC chemistry. After screening for optimal linker length and the amino acid composition of the peptide, compound 111 was identified as a potent bifunctional molecule disrupting the LIN28–let-7 interaction. The resulting novel class of chromenopyrazole–peptide conjugates showed improved properties in comparison with peptide-based probes. Therefore, proving the design strategy of the study and underlining the advantageous properties of conjugated entities over nonconjugated inhibitors2022-01-01T00:00:00ZComputational methodologies for DNA-encoded libraries
http://hdl.handle.net/2003/41229
Title: Computational methodologies for DNA-encoded libraries
Authors: Chines, Silvia
Abstract: Die Entdeckung von Arzneimitteln ist ein langwieriger und sehr kosten- und ressourcenintensiver Prozess. Daher wurden Screening-Technologien (physisch und virtuell) eingesetzt, um solche Prozesse zu optimieren und zu beschleunigen. DNA-kodierte Bibliotheken (DELs) haben sich in den letzten 30 Jahren als Alternative zum Hochdurchsatz-Screening herauskristallisiert, da sie zahlreiche Vorteile bieten. Die DEL-Technologie birgt jedoch drei große Herausforderungen: der Erhalt des DNA-Barcodes, die Abdeckung des chemischen Raums und die Identifizierung der Hits. Diese Faktoren werden durch die Größe der DNA-kodierten Bibliotheken und die daraus resultierende Menge der erzeugten Daten noch erschwert. Daher wurden chemoinformatische Ansätze verwendet, wie die KNIME-Analytics Platfrom. Im ersten Kapitel dieser Arbeit (Chemistry selection) wird ein Algorithmus vorgestellt, der den Raum der chemischen Reaktionen kartiert und sortiert, um Reaktionen auszuwählen, die potenziell für die DELSynthese verwendet werden können. Solche Reaktionen müssen Anforderungen erfüllen, welche die Integrität des DNA-Barcodes gewährleisten. Darüber hinaus beschreibt der Algorithmus die Reaktionen anhand spezieller molekularer Deskriptoren, die den Kern der Reaktion, d. h. den Teil der Reaktanten, der in Produkte umgewandelt wird, berücksichtigen. Auf der Grundlage solcher Deskriptoren wurden die Reaktionen geclustert, um den chemischen Reaktionsraum abzubilden. Aus den Clustern konnten Reaktionen erfolgreich auf DNA-kodierte Substrate angewendet werden. Im zweiten Kapitel (Building blocks selection) wurde die KNIME Analytics Platform eingesetzt, um die Bausteine (BBs) für die Bibliothekssynthese auszuwählen, mit dem Ziel, die strukturelle Vielfalt zu erhöhen. Virtuelle Bibliotheken konnten entsprechend der ausgewählten BBs und der etablierten DNA-kompatiblen Chemien aufgelistet werden. Die Reaktionen wurden mit Datenbanken arzneimittelähnlicher Verbindungen verglichen. Im dritten Kapitel (DNA-encoded library validation by molecular docking) wurden die Hit-Moleküle mit molekularer Docking-Software validiert. Drei Proteine wurden detailliert beschrieben und ihre jeweiligen Hits wurden als Liganden im Docking-Verfahren verwendet. Die Anwendung des Dockings als Validierungsmethode erwies sich als nützlich für die Bestätigung von Treffern, für die2022-01-01T00:00:00ZAmphiphile Block-Copolymere und eine Affinitäts-Aufreinigung für die DNA-kodierte Chemie
http://hdl.handle.net/2003/41206
Title: Amphiphile Block-Copolymere und eine Affinitäts-Aufreinigung für die DNA-kodierte Chemie
Authors: Götte, Katharina
Abstract: Die Kombination von molekularbiologischen Methoden, wie der genetischen Kodierung, Selektion, DNA-Amplifikation und DNA-Sequenzierung, mit der kombinatorischen organischen Chemie konnte große Sammlungen an Molekülen, sogenannte DNA-kodierte Bibliotheken, für die Identifikation von bioaktiven Molekülen ermöglichen. Dabei besteht eine große Herausforderung in der nur geringen Abdeckung des chemischen Raumes durch die notwendige Kompatibilität der verwendeten organischen Reaktionen mit dem DNA-Barcode. So können stark saure, oder oxidierende Bedingungen, sowie hohe Temperaturen zu einem Verlust der Integrität der genetischen Kodierung führen, wodurch eine Identifizierung von bioaktiven Molekülen erschwert wird. Zudem muss eine hohe Reinheit der Molekülbibliothek vorliegen, um die Identifikation falsch positiver Hits in Selektionsassays zu reduzieren. Um diese Herausforderungen anzugehen, wurden in dieser Arbeit amphiphile Block-Copolymere für die Synthese einer DNA-kodierten Substanzbibliothek verwendet, sowie eine neue Aufreinigungs-Methode etabliert und angewendet. Im ersten Teil dieser Arbeit wurden amphiphile Block-Copolymere eingesetzt, die in Wasser hydrophobe Reaktionsräume bilden und eine aktive katalytische Einheit, wie eine Sulfonsäure oder einen Palladium-Komplex, tragen. Dadurch sollte der hydrophile DNA-Barcode getrennt von dem reaktiven hydrophoben Reaktionsmedium vorliegen und somit eine Erweiterung des chemischen Raumes durch die Povarov-Reaktion, eine Boc-Entschützung, eine Pictet-Spengler-Reaktion, sowie eine Suzuki-Miyaura-Kreuzkupplung an DNA-Konjugaten ermöglicht werden. Da Proben aus chemischen Reaktionen an DNA-Konjugaten oftmals nicht ausreichend durch die verfügbaren Methoden aufgereinigt werden konnten, wurde im weiteren Verlauf dieser Arbeit eine neue Aufreinigung basierend auf einer dispersen Festphasenextraktion entwickelt. Schließlich konnte durch die etablierten Copolymer-vermittelten chemischen Reaktionen und der neuen Aufreinigungs-Methode eine 105.288-Mitglieder große DNA-kodierte Molekülbibliothek hergestellt und eine deutliche Verbesserung der Reinheit erzielt werden. Die starke Hydrophilie des DNA-Stranges birgt Einschränkungen in der Wahl des Lösungsmittels und somit der durchzuführenden chemischen Reaktion, da viele Reaktionen nur unter Ausschluss von Wasser erfolgreich ablaufen. Daher sollten in dem letzten Teil der Arbeit nicht-funktionalisierte amphiphile Block-Copolymere eingesetzt werden, um ein Vermischen von DNA-Konjugaten in organischen Lösungsmitteln zu ermöglichen. Diese Copolymer-DNA-Interaktion könnte eine neue Herangehensweise für die Durchführung von chemischen Reaktionen an DNA-Konjugaten in organischen Lösungsmitteln liefern.2022-01-01T00:00:00ZTargeting PRMT5 protein-protein interactions with peptidic modulators
http://hdl.handle.net/2003/41205
Title: Targeting PRMT5 protein-protein interactions with peptidic modulators
Authors: Krzyzanowski, Adrian
Abstract: Protein arginine methyltransferase 5 (PRMT5) is an important enzyme, methylating arginine residues of its substrates. PRMT5 is involved in a very large number of various cellular process, and has strong ties to a plethora of health disorders. PRMT5 mediates a number of protein-protein interactions (PPIs) with diverse adaptor proteins, such as the obligate binding partner MEP50, and adaptor proteins RioK1, pICln and COPR5. Modulation of PRMT5 PPIs appears as an attractive alternative to the inhibition of the active methylation site, giving an opportunity for significantly more precise targeting of the PRMT5 functionality in cells. This thesis describes pathways for development of peptidomimetics intended for the inhibition of various PPIs formed by PRMT5 or its adaptor protein MEP50. PPI between PRMT5 and MEP50 was investigated using available structural data to design inhibitory peptides. The compounds were synthesised and evaluated using biophysical and enzymatic assays. None of the synthesised compounds showed any activity in the employed assays. In the second part, the thesis describes the analysis of a potential PPI between SUZ12 and MEP50. A number of SUZ12 fragments spanning different protein regions were synthesised and tested for the direct interaction with the PRMT5-MEP50 complex. The analysis indicated that the short SUZ12 sequence CPWCTL is responsible for the interaction with the methyltransferase complex. Further analysis with the binding sequence and control proteins showed very strong non-specific interactions. In light of the obtained results any further peptidomimetic development based on the SUZ12 sequence was not pursued. The final chapters of the thesis focus on the PPIs between PRMT5 and adaptor proteins RioK1, pICln and COPR5. A consensus sequence GQF[D/E]DA[D/E], identified in the terminal regions of the adaptor proteins, was determined to be responsible for mediating the interactions with PRMT5. Biophysical assays and X-ray crystallography allowed to characterise the binding interface and revealed that the consensus sequence binds to the non-catalytic TIM barrel domain of PRMT5. The biophysical data were then used for the design of highly stable macrocyclic PRMT5 adaptor protein interaction inhibitors. Optimisation of the molecular structure resulted in a potent cyclic PRMT5 binder (KD = 89 nM). The developed molecule could inhibit the PPI between PRMT5 and full length pICln when tested in vitro (IC50 = 654 nM). Examination of the compound activity in cellular lysates revealed a surprising selectivity towards the inhibition of the PRMT5 interactions with RioK1 over the interactions with pICln. The developed compound did not supress the enzymatic activity of the methyltransferase and did not inhibit the interaction with MEP50.2022-01-01T00:00:00ZInvestigating a role for EDI3 in tumor growth and metastasis in breast cancer using a doxycycline-inducible knockdown system
http://hdl.handle.net/2003/41196
Title: Investigating a role for EDI3 in tumor growth and metastasis in breast cancer using a doxycycline-inducible knockdown system
Authors: Glotzbach, Annika
Abstract: Metastasis remains a major problem for tumor therapy. In endometrial and ovarian cancer, metastasis and worse survival was found to be associated with elevated EDI3 (GPCPD1; GDE5; GDPD6) expression in primary tumors. EDI3 is a glycerophosphodiesterase which cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P) and is therefore considered one of the key enzymes involved in choline metabolism. Altered choline metabolism is a recognized metabolic hallmark of cancer and was reported in breast, ovarian, and prostate cancers. Previously, in vitro studies revealed that silencing EDI3 transiently in various breast cancer cell lines resulted in altered choline metabolism and impaired cellular migration, attachment, and spreading. However, stable constitutive EDI3 knockdown led to compensation of metabolite levels over time, which was accompanied by a loss of the migration phenotype. Therefore, in the present work a doxycycline (Dox) inducible EDI3 knockdown system was established in luciferase-expressing ER-HER2+ breast cancer cells, which reduces compensatory effects and allows to investigate EDI3 in tumor growth and metastasis in vivo. To create cell lines in which EDI3 is inducibly silenced, stable luciferase-expressing HCC1954 cells were generated and subsequently transduced with lentiviral particles, which resulted in three different Dox-inducible EDI3 knockdown cell lines containing independent EDI3-targeting shRNA oligos. Dox treatment led to a time and dose dependent decrease in EDI3 RNA and protein expression. Mass spectrometry analyses revealed that induced EDI3 knockdown also led to dose dependent alterations in endogenous choline metabolites and phospholipid levels. Using various in vitro assays, it could be shown that EDI3 knockdown resulted in significant reduction in colony formation and proliferation, processes which are relevant in the formation of metastasis. Furthermore, EDI3 silencing rendered cells more susceptible towards anoikis. However, Dox-induced EDI3 knockdown had only little effect on adhesion and no effect on migration. To investigate EDI3’s role in tumor growth and metastasis in vivo, different tumor models were established in immunodeficient mice. The subcutaneous tumor model showed no significant effect on primary tumor growth. However, in a mouse model for peritoneal metastasis, luminescence imaging revealed lower signals indicative of less metastasis formation in the EDI3 knockdown condition. Furthermore, it could be shown that silencing EDI3 was associated with reduced tumor burden, less ascites fluid and longer survival time. Altogether, this thesis provides, for the first time, in vivo evidence that supports a role for EDI3 in metastasis formation, which further emphasizes the importance of choline and glycerophospholipid metabolism in this process.2022-01-01T00:00:00ZDifferentiation of distinct mesoderm subtypes from regulatory interactions between FGF and BMP signaling
http://hdl.handle.net/2003/41184
Title: Differentiation of distinct mesoderm subtypes from regulatory interactions between FGF and BMP signaling
Authors: Gattiglio, Marina
Abstract: Distinct mesodermal precursors arise during gastrulation in a precise temporal and spatial manner. In the mouse, the cells differentiating earlier in a more proximal position form the precursors for the heart and vasculature, followed in a more distal position by precursors of the urogenital system and then the musculoskeletal system. Mesoderm patterning is controlled by signals such as BMP4, produced by the extraembryonic ectoderm, and FGFs and WNTs produced by the epiblast itself. How do these signals interact in this process and whether cell-cell crosstalk can influence mesoderm patterning is still largely unknown.
Here I address this question with a 2D cell culture model that employs primed pluripotent stem cells resembling mouse epiblast cells and differentiates them with precise concentrations of different signaling molecules in a chemically defined medium. By titrating BMP4, I observe that high concentrations of BMP4 promote the expression of proximal mesoderm markers, while intermediate to low concentrations favour distal and pan-mesoderm markers. The opposite effect is seen with a titration of FGF. A scRNAseq analysis revealed that endogenous FGF signaling lead to the differentiation of heterogeneous mixtures of cell types, and that their proportions were affected by the dose of exogenous FGF. Furthermore, higher doses of exogenous FGF reduced the expression of BMP ligands in the cells, and promoted the expression of selected WNT ligands and endogenous FGFs, indicating the existence of a positive FGF feedback loop. Cells associated with high FGF signaling levels were also found to be spatially clustered in the cultures, suggesting that this loop could generate coherent clusters of cells with discrete identities in a cell population. Based on these findings, I propose a molecular mechanism for the FGF feedback loop. Together, these results indicate that the patterning of the mesoderm is dependent on a combination of external signaling and cell-cell crosstalk.; Während der Gastrulation entstehen distinkte mesodermale Vorläuferzellen in einer präzisen zeitlichen und räumlichen Abfolge. In der Maus bilden Zellen, die früh aus proximalen Positionen differenzieren, die Vorläufer von Herz und Gefäßsystem, während später aus distalen Positionen Vorläufer des urogenitalen Systems und anschließend des Bewegungsapparats gebildet werden. Die Identität mesodermaler Zellen wird durch Signale wie BMP4, die vom extraembryonalen Ektoderm produziert werden, sowie von FGFs und WNTs, die vom Epiblast selbst produziert werden, kontrolliert. Wie diese Signale interagieren und ob Zell-Zell-Kommunikation die Mesodermbildung beeinflusst, ist größtenteils unbekannt.
Um diese Frage zu beantworten verwende ich ein 2D-Zellkulturmodell aus epiblastartigen Mausstammzellen, die mit verschiedenen Konzentrationen bestimmter Signalmoleküle in einem chemisch definierten Medium differenziert werden. BMP4-Titrierung zeigte, dass hohe Konzentrationen von BMP4 die Expression proximaler Mesodermmarker unterstützen, während mittlere bis geringe Konzentrationen distale und panmesodermale Marker begünstigen. Das Gegenteil wird durch FGF-Titrierung ersichtlich. Die Analyse von scRNAseq-Daten zeigte, dass endogene FGF-Signale zur Differenzierung heterogener Zelltypen führen, deren Verhältnis von der Dosis von exogenem FGF abhängt. Zudem reduzierten höhere Dosen exogenen FGFs die Expression von BMP-Liganden in den Zellen und unterstützten die Expression einiger bestimmter WNT-Liganden und endogenen FGFs, was die Existenz eines positiven FGF-Feedback-Loops impliziert. Zellen, die mit einem hohen FGF-Signal-Level assoziiert sind, waren außerdem innerhalb der Kultur räumlich geclustert, was die Vermutung nahelegt, dass der FGF-Feedback-Loop kohärente Zellcluster mit diskreten Identitäten innerhalb einer Zellpopulation generiert. Basierend auf diesen Erkenntnissen schlage ich einen molekularen Mechanismus für den FGF-Feedback-Loop vor. Zusammengenommen zeigen diese Ergebnisse, dass die mesodermale Identität in Abhängigkeit einer Kombination von externen Signalen und Zell-Zell-Kommunikation bestimmt wird.2022-01-01T00:00:00ZStructural insights into muscle organisation by electron cryo-tomography
http://hdl.handle.net/2003/41176
Title: Structural insights into muscle organisation by electron cryo-tomography
Authors: Wang, Zhexin
Abstract: Movement is the essence of life in the animal realm. Skeletal muscle is an essential tissue specialised for movement. Muscle cells are multi-nucleated cells containing bundles of myofibrils, which are segmented into the smallest contractile units, named sarcomeres. While sarcomeres are known to contain thin (actin) and thick (myosin) filaments, the detailed architecture, especially the high-resolution and 3-dimensional (3D) information, remains obscure. In this thesis, I obtained the first high-resolution 3D pictures of the sarcomere using cryo-focused ion beam milling (cryo-FIB-milling) and electron cryo-tomography (cryo-ET). The sarcomere organisation highlights a molecular plasticity which ensures efficient muscle contraction in different environments. Furthermore, from these native 3D images, I determined the first structures of the ruler protein, nebulin, to a resolution of 4.5 Å, which establishes the molecular basis for its functions in thin filament stabilisation, length control and myosin-binding regulation. The in situ structures also revealed a double-head conformation of myosin that reveals inherent variability to increases myosin’s capability for binding to the thin filaments. Collectively, my thesis research provides unique insights into muscle structures that allow improved dynamic modelling of muscle contraction and muscle diseases. This research also establishes a new cryo-FIB-ET approach for structurally characterising muscle components in different types of muscles and diseased states.2022-01-01T00:00:00ZRecursion quantification analysis reveals a cellular morphodynamics - ERK activity recursive relationship
http://hdl.handle.net/2003/41175
Title: Recursion quantification analysis reveals a cellular morphodynamics - ERK activity recursive relationship
Authors: Campos-Medina, Manuel
Abstract: One of the enigmas of cellular biology is the process by which eukaryotic cells,
abandoning their unitary role, organize to form complex systems of organs. Despite
the details of such pattern emergence process are yet to be elucidated, there is a
significant degree of coordination among adjacent cells. For decades, several proteins
have been monitored to track cellular communication. Although advancements have
been made regarding our understanding of the internal cellular signaling network,
there is no clear answer regarding how cells can communicate. One of the most
studied families of proteins is the Extracellular-signal-regulated-kinases. Due to its
myriad interactions and influence over most major cellular processes, it has been
established as a central node in the cellular signaling network. However, the number
of studies regarding its dynamic behavior is limited. Although its connection with
cellular morphodynamics has also been well-documented, there is no consensus on
the time scale of their relationship.
By studying ERK's dynamics upon growth factor stimulation, we found that the
local cellular environment influences ERK's activation tendencies. Nonetheless, this
effect is more significant at low growth factor concentrations. We employed a nonlinear
dynamics multidimensional analysis to explore the existence of recurring
patterns within ERK dynamics and cellular morphodynamics. We focused on the highfrequency
components of each of these dynamics during the basal state of cells and
after either EGF or HRG stimulation. After cells were exposed to cell-fate-decision
induing dosages of growth factors, our analysis showed an increase in the persistence
of ERK dynamics and cellular morphodynamics recurrence patterns. Furthermore,
we found that the recurrences that exist within ERK dynamics and cellular
morphodynamics occur at similar time scales. Moreover, our results showed that these
two dynamic processes are intrinsically related, and their relationship is not modified
upon growth factor stimulation.
By extending the recurrence analysis to neighboring cells' dynamic behavior,
we corroborated our findings on the influence of the local cellular environment on ERK
dynamics and cellular morphodynamics. ERK dynamics in neighboring cells kept their
communication pattern after EGF stimulation. However, cellular morphodynamics
between neighboring cells synchronized only after stimulation with low concentrations
of EGF.
Our work sets the framework for conceiving ERK dynamics and cellular
morphodynamics as inseparable processes. It also sheds light on conceptualizing
cellular communication as a process that could occur via physiological concentrations
of EGF among MCF-7 cells.; Eines der Rätsel der Zellbiologie ist der Prozess, durch den sich eukaryotische
Zellen unter Aufgabe ihrer einheitlichen Rolle zu komplexen Organsystemen
organisieren. Obwohl die Einzelheiten eines solchen Musterbildungsprozesses noch
nicht geklärt sind, gibt es ein erhebliches Maß an Koordination zwischen benachbarten
Zellen. Seit Jahrzehnten werden verschiedene Proteine überwacht, um die zelluläre
Kommunikation zu verfolgen. Obwohl unser Verständnis des internen zellulären
Signalnetzwerks Fortschritte gemacht hat, gibt es keine klare Antwort darauf, wie
Zellen kommunizieren können. Eine der am meisten untersuchten Proteinfamilien ist
die der extrazellulären signalregulierten Kinasen. Aufgrund ihrer unzähligen
Interaktionen und ihres Einflusses auf die meisten wichtigen zellulären Prozesse hat
sie sich als zentraler Knotenpunkt im zellulären Signalnetzwerk etabliert. Die Zahl der
Studien zu ihrem dynamischen Verhalten ist jedoch begrenzt. Obwohl sein
Zusammenhang mit der zellulären Morphodynamik gut dokumentiert ist, gibt es keinen
Konsens über die Zeitskala ihrer Beziehung.
Bei der Untersuchung der ERK-Dynamik nach Stimulation durch
Wachstumsfaktoren haben wir festgestellt, dass die lokale zelluläre Umgebung die
Aktivierungstendenzen von ERK beeinflusst. Dieser Effekt ist jedoch bei niedrigen
Konzentrationen des Wachstumsfaktors stärker ausgeprägt. Wir setzten eine
multidimensionale Analyse der nichtlinearen Dynamik ein, um die Existenz
wiederkehrender Muster innerhalb der ERK-Dynamik und der zellulären
Morphodynamik zu untersuchen. Wir konzentrierten uns auf die hochfrequenten
Komponenten jeder dieser Dynamiken während des Grundzustands der Zellen und
nach EGF- oder HRG-Stimulation. Nachdem die Zellen einer den Zellzustand
beeinflussenden Dosis von Wachstumsfaktoren ausgesetzt wurden, zeigte unsere
Analyse eine Zunahme der Persistenz der ERK-Dynamik und der zellulären
Morphodynamik-Rezidivmuster. Darüber hinaus stellten wir fest, dass die
Wiederholungen innerhalb der ERK-Dynamik und der zellulären Morphodynamik auf
ähnlichen Zeitskalen auftreten. Darüber hinaus zeigten unsere Ergebnisse, dass diese
beiden dynamischen Prozesse eng miteinander verbunden sind und ihre Beziehung
durch die Stimulation mit Wachstumsfaktoren nicht verändert wird.
Indem wir die Rekurrenzanalyse auf das dynamische Verhalten benachbarter
Zellen ausdehnten, konnten wir unsere Erkenntnisse über den Einfluss der lokalen
zellulären Umgebung auf die ERK-Dynamik und die zelluläre Morphodynamik
untermauern. Die ERK-Dynamik in benachbarten Zellen behielt ihr
Kommunikationsmuster nach der EGF-Stimulation bei. Die zelluläre Morphodynamik
zwischen benachbarten Zellen synchronisierte sich jedoch nur nach Stimulation mit
niedrigen EGF-Konzentrationen.
Unsere Arbeit bildet den Rahmen, um die ERK-Dynamik und die zelluläre
Morphodynamik als untrennbare Prozesse zu begreifen. Sie wirft auch ein Licht auf
die Vorstellung, dass zelluläre Kommunikation ein Prozess ist, der durch
physiologische EGF-Konzentrationen zwischen MCF-7-Zellen stattfinden könnte.2022-01-01T00:00:00ZStructural basis for specific inhibition of the deubiquitinase UCHL1
http://hdl.handle.net/2003/41164
Title: Structural basis for specific inhibition of the deubiquitinase UCHL1
Authors: Grethe, Christian; Schmidt, Mirko; Kipka, Gian-Marvin; O’Dea, Rachel; Gallant, Kai; Janning, Petra
Abstract: Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.2022-10-10T00:00:00ZReconstitution of RhoGTPase membrane loading
http://hdl.handle.net/2003/41098
Title: Reconstitution of RhoGTPase membrane loading
Authors: Armstrong, Michael
Abstract: The polarisation of membrane-bound signalling molecules that control the actin cytoskeleton, such as Rho-type GTPases and phosphoinositides is essential for the spatial organisation of cell shape. However, the ubiquitous solubilisation factor RhoGDI (Guanosine Dissociation Inhibitor) sequesters the majority of RhoGTPases in the cytosol, away from the membrane. Starting from defined complexes of RhoGTPases and RhoGDI, we investigate how RhoGTPases load onto membranes.
To this end, we reconstituted the recruitment of the RhoGTPase Cdc42 to supported lipid bilayers and followed this process by multi-colour single molecule imaging. This allowed us to investigate of the effects of several suggested regulators of Cdc42 recruitment. These included regulators catalytic cycle such as activating guanosine exchange factors (GEFs), the biochemical composition and characteristics of the membrane such as charge or lipid composition, and the use of cell lysate to probe for a still unidentified GDI dissociation factor (GDF).
By capturing these events as a high time resolution, it was possible to not only understand what modulates Cdc42 recruitment, but also to decipher at what point during the recruitment process GDI dissociated.
Using reconstitution along with powerful TIRF microscopy and the development of single molecule tracking approaches we showed that RhoGTPase separate from their solubilisation factor RhoGDI prior binding to the membrane. Furthermore, once separated from RhoGDI, it is the composition of the membrane itself which regulates the binding frequency of RhoGTPases at the membrane. We go on to show that, on a supported lipid bilayer, the GEF ITSN is incapable of promoting recruiting of Cdc42 alone, nor the Cdc42:GDI complex suggesting that the catalytic and spatial cycles of the RhoGTPases not coupled in the process of RhoGTPase membrane recruitment.
Finally, through the inclusion of cell lysate in our reconstitutions and in contrast to assumptions in the field, we observe no evidence for an essential active RhoGDI displacement factor (GDFs) that facilitates RhoGDI removal.2022-01-01T00:00:00ZOptochemical control of DNA methylation erasers and readers enables kinetic insights into their domain-dependent interplay
http://hdl.handle.net/2003/41094
Title: Optochemical control of DNA methylation erasers and readers enables kinetic insights into their domain-dependent interplay
Authors: Lin, Tzu-Chen
Abstract: 5-Methylcytosine (5mC) is a central epigenetic mark of mammalian DNA. It mainly occurs in cytosine-guanine (CpG) dinucleotides and is recognized competitively by methyl-CpG binding domain (MBD) proteins and ten-eleven-translocation (TET) dioxygenases, which act as methylation readers and erasers to mediate regulatory chromatin crosstalk and epigenome editing, respectively. The dynamic reader-eraser interplay at their common substrate is therefore highly regulated for a coherent transcriptional program. However, mechanistic insights of their interplay are hampered by a lack of suitable methodology to control their activities in living cells.
This work employs light-activatable human TET1 and MBD1 to enable precise temporal control of enzymatic oxidation activity or substrate recognition. Light activation is achieved by genetic encoding of a photocaged serine that can be co-translationally incorporated at critical protein sited in mammalian cells. On the one hand, monitoring the TET1-catalyzed 5mC oxidation kinetics in vivo reveals a multifaceted domain-dependent modulation by MBD1. While the MBD domain of MBD1 negatively regulates TET1 oxidation kinetics and dominates the interplay by competing for the 5mC substrates, the third Cys-x-x-Cys (CXXC3) domain of MBD1 contrarily modulates TET1 activity by binding to nonmethylated CpGs. Intriguingly, the transcriptional repressor domain (TRD) does not influence 5mC oxidation kinetics by TET1. On the other hand, studies with light-activatable MBD1 indicate a domain-dependency of cellular mCpG binding kinetics. Depriving the nonmethylated CpG affinity of the CXXC3 domain enhances binding kinetics, whereas the absence of the TRD domain results in decreased binding kinetics. Moreover, the light-activatable MBD1 can further unveil the mechanism of MBD1-TET1 interplay by uncoupling the process from prior binding events of MBD1. Collectively, this work enables first kinetic insights into the domain-dependent interplay of methylation readers and erasers in the natural chromatin context and provides novel tools to unravel the dynamic chromatin regulation program.2022-01-01T00:00:00ZStem cell-based phenotypic screening and characterization of novel BMP mimetics
http://hdl.handle.net/2003/41067
Title: Stem cell-based phenotypic screening and characterization of novel BMP mimetics
Authors: Weßeler, Fabian
Abstract: The identification of selective small molecule cytokine mimetics and signaling activators holds great promise for numerous applications in biomedicine as they overcome the typical drawbacks of physiological peptide- or protein-based ligands. Yet, the development of such modalities remains a challenging task in drug discovery. In this thesis, a phenotypic, target-agnostic, high-throughput screening assay is presented that probes Bone Morphogenetic Protein (BMP) signaling during mesodermal patterning of murine embryonic stem cells. This approach represents a novelty in BMP activator identification by harnessing embryonic development in vitro, hence potentially expanding the druggable space of BMP signaling activators. During mesoderm specification, BMP signaling can be temporally discriminated from Transforming Growth Factor- (TGF)-driven stages of cardiac differentiation. This selective and authentic orchestration of BMP signaling cues can be recapitulated for the discovery of genuine BMP activator- or potentiator chemotypes. Here, a robust hit validation workflow has been devised, including the orthogonal assessment of BMP activation during osteoblastogenesis as well as BMP-dependent reporter assays. Proof-of-concept is demonstrated from screening of nearly 7,000 chemically diverse compounds, yielding 2,3-disubstituted 4H-chromen-4-ones as a new potent BMP activating chemical modality.
Chromenone 1 enhanced osteogenic differentiation and mineralization in vitro. Structure-activity relationship (SAR) studies with 29 different Chromenones revealed pharmacophoric features relevant to BMP activity and provided a valuable toolkit of active and structurally closely related inactive derivatives. Interestingly, mechanistic studies suggested that Chromenone 1 enhances canonical nuclear BMP-Smad signaling outputs through an unparalleled, kinase-independent, negative TGF-Smad feedback loop. This is in sharp contrast to the reported BMP sensitizer PD407824, revealing novel BMP potentiator biology as desired from a conceptionally new morphogenic and phenotypic drug discovery approach.
An additional screening of 1,408 known bioactive compounds eventually furnished the triazolo[1,5 c]quinazoline CGS-15943 as a novel and unique BMP activating chemotype that exhibited highest selectivity among all profiled and characterized BMP modulators in the present work. Its capacity to amplify BMP signaling during embryogenesis could be further substantiated in vivo during zebrafish development (i.e., ventralization phenotypes). Target deconvolution studies unraveled novel targets of CGS-15943 with a pronounced potency for CK1- and --isoforms. Mechanistically, CGS-15943 amplifies osteogenic BMP signaling outputs through a dual targeting of CK1/ and PI3K (p110) isoforms by enhancing BMP signaling independent of a TGF-feedback.
Together, the presented work underscores proof-of-concept for a novel phenotypic drug discovery strategy to identify unique BMP signaling modulators. Two new chemotypes ultimately expanded the druggable space of osteogenic BMP activators and sensitizers that may serve as valuable tools for various applications in (stem) cell biology and regenerative medicine. Notably, this work might spur future translation to even more complex 3D-gastruloid and organoid systems.2021-01-01T00:00:00ZMolecular insights into Bacillus anthracis anthrax lethal complex
http://hdl.handle.net/2003/41056
Title: Molecular insights into Bacillus anthracis anthrax lethal complex
Authors: Antoni, Claudia
Abstract: Bacillus anthracis secretes the major virulent factor anthrax complex toxin, which is responsible for high mortality in mammals. This tripartite complex comprises three soluble proteins: the protective antigen (PA) and two enzymes, the lethal factor (LF) and edema factor (EF). After proteolytic activation by host proteases, PA oligomerizes into octamers and/or heptamers on the cell surface and the LF as well as EF bind competitively to the pre-pore oligomer surface. After a receptor-mediated endocytosis, the pre-pore complex rearranges in a narrow pore channel that delivers the two unfolded enzymes into the cytosol. There, the LF and EF lead to the inactivation of the mitogen-activated protein kinases (MAPKKs) and the dysregulation of the level of cellular cAMP, respectively.
So far, only fragmented information about the heptameric PA in complex with LF are available. Particularly, complex organization and subunits interaction, remain unclear. In this work, I apply cryo-electron microscopy (cryo-EM) to solve the structure of the fully loaded complex of PA with LF (PA7LF3) in the pre-pore conformation. Our data demonstrate features of LF molecules assembling in the PA7LF3 complex, showing for the first time three different complex combinations, namely PA7LF2+1A PA7LF2+1A’ and PA7LF2+1B. These complexes vary on the 3D LF organization on the PA. Our structural analysis defines LF-LF and PA-LF interactions revealing a new potential interaction
interface at the C-terminus of LFs. Finally, we demonstrate an intermediate conformation of LF which was not previously predicted. These results show for the first time that the PA7LF3 can generate three different complex states. This work adds another milestone on the intricated mechanism of the anthrax complex translocation, which culminates in the release of LF and EF into the host cytoplasm.; Das Bakterium Bacillus anthracis sorgt durch die Sekretierung seines Toxins, dem Anthrax Komplex, der den Hauptteil des virulenten Faktors ausmacht, für eine hohe Mortaliät in allen Säugern. Dabei besteht der Komplex aus den folgenden drei löslichen Proteinen: dem schützenden Antigen (protective antigen, PA) sowie zwei Enzymen, dem letalen Faktor (lethal factor, LF) und dem Edema Faktor (edema factor, EF). Nach einer proteolytischen Aktivierung des Komplexes von Wirtsproteasen oligomerisiert das PA an der Zelloberfläche zu Octameren und/oder Heptameren. Dabei binden der LF sowie der EF kompetitiv an die Zelloberfläche und bilden so einen Prä-Porzustand. Die Bindung zu einem Rezeptor initiiert weiter die Endozytose, nach welcher der Prä-
Porzustand zu einem schmalen Porenkanal umgelagert wird und die beiden ungefalteten Enzyme in das Zytosol gelassen werden. Nach der Rückfaltung der Enzyme in deren nativen und enzymatisch aktiven Zustand im Zytosol sorgt der LF für die Inaktivierung der mitogen-activierenden Protein Kinasen (MAPKKs) und der EF zu einem unregelmäßigen Level an zellulärem cAMP.
Bis Heute gibt es nur limitierende Informationen zu dem kompletten PA7 Komplex mit
dem LF (PA7LF3), wobei insbesondere die Komplexformierung sowie die intramolekularen Interaktionen der einzelnen Untereinheiten noch unklar sind. In dieser Arbeit habe ich mithilfe der Kryo-Elektronenmikroskopie (cryo-EM) die Struktur des gesamten Komplexes im Prä-Porenzustand bestehend aus PA und LF (PA7LF3) gelöst, wodurch die intermolekularen Interaktionen zwischen LF-LF sowie LF-PA näher untersucht werden konnten und noch unbekannte Komplexformierungen beobachtet werden konnten. Hier konnten insgesamt drei neue Kombinationsmöglichkeiten des
Komplexes beobachtet werden: PA7LF(2+1A), PA7LF(2+1A’) und PA7LF(2+1B). Die Strukturanalyse dieser neuen Komplexformierungen zeigten zudem noch unbekannte Konformationen von LF im gebunden Zustand des heptameren PA, die entlang der gesamten Kette Interaktionen aufzeigte. Zudem wies ausschließlich eine der drei LF Moleküle in dem PA α-clamp-gebunden Zustand die offene Konformation auf, in der eine erweiterete Flexibilität der C-terminalen Domäne beobachtet wurde. Die beiden anderen LFs hingegen, zeigen eine neue Konformation und befinden sich in einem
sogenannten Intermediären Zustand zwischen der Toxinassemblierung und der Translokation. Durch die Präsenz beider Konformationszustände der LFs in dem PA7LF3 Komplex kann von einem neuen Modell der Translokation ausgegangen werden, in dem die LFs in Abhängigkeit ihrer Konformation durch den PA7 Kanal in die Wrtszelle transloziert werden.2022-01-01T00:00:00ZApplication of the cell painting assay for pseudo-natural product projects
http://hdl.handle.net/2003/41055
Title: Application of the cell painting assay for pseudo-natural product projects
Authors: Zinken, Sarah
Abstract: Considering that natural products (NPs) underwent evolutionary selection to bind to their biological targets and therefore represent biologically prevalidated chemical matter, they can serve as a valuable source of inspiration. In the pseudo-NP (PNP) approach, NP fragments are combined by means of chemical methodologies in new arrangements that are not observed in nature. PNPs are designed to share the NP fragments’ high probability to bind to macromolecular targets but also may explore novel areas of biological space as they are structurally distinct from NPs. Phenotypic screening methods, particularly the cell-painting assay (CPA), have been investigated as powerful tools for the biological evaluation of PNPs since they may have unexpected bioactivities. Herein, a PNP class derived from a quinine- and azaindole-fragments was proven to inhibit the autophagy signalling pathway. Comparison of its morphological profile from the CPA to reference compound profiles with annotated bioactivity hinted towards a kinase target. Further investigation proved this hypothesis and identified VPS34 kinase as a potential autophagy-related target. This study highlighted the impact that the CPA can have on the target identification of bioactive PNPs. CPA analysis of diverse PNP compound classes derived from a small fragment subset, allowed the investigation of fragment combinations on morphological profiles. It was shown that fragments can either have a dominating or non-dominating effect on the morphological activity of PNPs that could also be proven in behaviour predictions. This led to the suggestion to use non-dominating fragments to design PNPs with unique and potentially novel bioactivities. Taking inspiration from previous PNP examples, a novel chromane- and 4H-quinoline-derived compound class was designed. The development of a suitable synthetic route resulted in a chromaline compound library that was subsequently tested in the CPA. Comparison with the inspiring PNP classes (chromopynone and pyrroquinoline) revealed, that both fragments were non-dominating and therefore suitable for the design of unique PNPs. Furthermore, the novel chromaline compound class showed high chemical and morphological diversity. Overall, design strategies with the guiding principle of PNPs were employed to generate compound libraries that were evaluated and biologically characterised by the cell painting assay.2022-01-01T00:00:00ZDesign and synthesis of Nrf2-derived hydrocarbon stapled peptides for the disruption of protein-DNA-interactions
http://hdl.handle.net/2003/41010
Title: Design and synthesis of Nrf2-derived hydrocarbon stapled peptides for the disruption of protein-DNA-interactions
Authors: Wiedemann, Bianca; Kamps, Dominic; Depta, Laura; Weisner, Jörn; Cvetreznik, Jana; Tomassi, Stefano; Gentz, Sascha; Hoffmann, Jan-Erik; Müller, Matthias P.; Koch, Oliver; Dehmelt, Leif; Rauh, Daniel
Abstract: Misregulation and mutations of the transcription factor Nrf2 are involved in the development of a variety of human diseases. In this study, we employed the technology of stapled peptides to address a protein-DNA-complex and designed a set of Nrf2-based derivatives. Varying the length and position of the hydrocarbon staple, we chose the best peptide for further evaluation in both fixed and living cells. Peptide 4 revealed significant enrichment within the nucleus compared to its linear counterpart 5, indicating potent binding to DNA. Our studies suggest that these molecules offer an interesting strategy to target activated Nrf2 in cancer cells.2022-06-22T00:00:00ZDesign, Synthese und Evaluation Mutanten-selektiver Inhibitoren zur Adressierung onkogener KRas-Varianten
http://hdl.handle.net/2003/40997
Title: Design, Synthese und Evaluation Mutanten-selektiver Inhibitoren zur Adressierung onkogener KRas-Varianten
Authors: Goebel, Lisa
Abstract: Ras-Proteine fungieren als molekulare Schalter in der Signaltransduktion essentieller zellulärer Prozesse, indem sie zwischen einem inaktiven GDP-gebundenen und einem aktiven GTP-gebundenen Zustand wechseln. Onkogene Ras-Mutationen, die in etwa 25 % aller humanen Krebsarten vorkommen und zu einer Fehlregulation des switch Mechanismus führen sind daher attraktive Zielstrukturen in der Präzisionsmedizin. Trotz jahrzehntelanger intensiver Forschung nach Entdeckung der Ras-Onkogene im Jahre 1981 waren Versuche, Ras gezielt zu adressieren, weitestgehend erfolglos und Ras-Proteine galten lange Zeit als nicht adressierbar (engl.: undruggable). Neue Hoffnung ergab sich jedoch im Jahr 2013 durch die erfolgreiche gezielte Adressierung der G12CMutation von Ras durch Kevan Shokat. Neben kovalenten Inhibitoren, die innerhalb der switch-II-Tasche irreversibel an die G12C-Mutation binden und deren Optimierung schließlich im Mai 2021 zur Zulassung des ersten KRasG12C-Inhibitors Sotorasib (Amgen) für die Behandlung von nicht-kleinzelligem Lungenkrebs (NSCLC) führte, wurden zudem Nukleotid-kompetitive Inhibitoren entwickelt, die ebenfalls kovalent an KRasG12C binden können. Strategien, die eine direkte Konkurrenz zur Nukleotidbindung beinhalten, wurden ursprünglich aufgrund der hohen Affinität von GDP/GTP für Ras und der hohen zellulären Nukleotid-Konzentrationen verworfen, erlangten jedoch durch die Kombination mit einem kovalenten Wirkmechanismus der Inhibitoren erneut Aufmerksamkeit im akademischen Umfeld. Die dabei am β-Phosphat der GDP-Derivate eingeführten elektrophilen Gruppen führten jedoch zu einer dramatischen Reduzierung der reversiblen Bindungsaffinität der Nukleotidderivate, da wichtige Wechselwirkungen mit dem Protein und dem Mg2+-Ion verloren gehen. Um diese Limitierung zu überwinden und diesen Ansatz der kovalenten Nukleotid-kompetitiven Inhibitoren an onkogene KRas-Varianten mit Cysteinen im P-loop (KRasG12C und KRasG13C) anzupassen, wurden im Rahmen dieser Arbeit mittels strukturbasiertem Wirkstoffdesign Nukleotidanaloga synthetisiert, die einen Thiolreaktiven Linker an den 2‘,3‘-OH-Gruppen der Ribose für eine kovalente Proteinmodifikation tragen. Anhand von massenspektrometrischen Experimenten konnte gezeigt werden, dass die dargestellten Nukleotidderivate selektiv an KRasG13C binden, ohne dabei KRasG12C sowie KRasWT zu adressieren. Mittels einer detaillierten kinetischen Charakterisierung wurden anschließend die Affinitäten der Nukleotidanaloga gegenüber KRas im Vergleich zu den unmodifizierten Nukleotiden bestimmt. Neben der Bestimmung der Kinetik der Nukleotidassoziation (kon) mit einer bereits etablierten stopped-flow Methode wurde im Rahmen dieser Arbeit zudem ein HPLC-basierter Assay zur Evaluierung der relativen Affinitäten der Nukleotidanaloga entwickelt. Die Affinitäten der dargestellten Nukleotidderivate sind dabei mit denen der unmodifizierten Nukleotide vergleichbar, sodass der eingeführte Linker keinen Einfluss auf die reversible Wechselwirkung und die Affinität hat, was bei einem Nukleotid-kompetitivem Ansatz von maßgeblicher Bedeutung ist. Im Rahmen dieser Arbeit konnte zudem gezeigt werden, dass bei KRasG13C, welches eine drastisch erhöhte intrinsische Nukleotidaustauschrate im Vergleich zum WT besitzt, infolge einer kovalenten Proteinmodifikation der SOS-katalysierter Nukleotidaustausch verhindert und kovalent modifiziertes KRasG13C im inaktiven Zustand stabilisiert wird. Eine gesteigerte GTP-Hydrolyse in Gegenwart von GAPs konnte für kovalent modifizierte KRasG13C im Vergleich zu KRasWT nicht beobachtet werden, die intrinsische GTP-Hydrolyse ist hingegen vergleichbar mit der von KRasWT. Die in dieser Arbeit generierten Komplexkristallstrukturen von KRasG13C-edaGDP und KRasG13C-bdaGDP ermöglichten darüber hinaus einen detaillierten Einblick in den Bindemodus der Nukleotidderivate und bestätigten den erwarteten Bindemodus sowie die kovalente Bindungsknüpfung an Cys13. Aufgrund der limitierten Verfügbarkeit von KRasG13C-abhängigen Krebszelllinien wurden zunächst erfolgreich artifizielle KRas-abhängige Zelllinien, darunter NIH-3T3 sowie Ba/F3-Zellen generiert, die in Zukunft für die Charakterisierung mutantenselektiver KRas-Inhibitoren genutzt werden können. Für die zelluläre Charakterisierung der in dieser Arbeit dargestellten Nukleotidanaloga, die aufgrund ihres anionischen Charakters nicht die Zellmembran überwinden können, wurden verschiedene Strategien für den Transfer in Zellen getestet, unter anderem ein NTP-Transporter vermittelter Transfer sowie die Elektroporation. Durch Elektroporation rekombinanter KRas-Proteine in Zellen konnte schließlich gezeigt werden, dass bei der Verwendung von kovalent modifiziertem KRasG13C eine onkogene Signalweiterleitung in vivo inhibiert wird, was die Möglichkeit des Einsatzes von kovalenten Nukleotid-basierten Inhibitoren bei KRasG13C-getriebenem Krebs verdeutlicht.2022-01-01T00:00:00ZDevelopment of methods toward the synthesis of novel bioactive natural product-like scaffolds
http://hdl.handle.net/2003/40996
Title: Development of methods toward the synthesis of novel bioactive natural product-like scaffolds
Authors: Davies, Caitlin
Abstract: The complexity and inherent biological relevance of natural products (NPs) are key characteristics that have made them a constant source of inspiration in the development of novel bioactive compounds. However, NPs alone cannot solve the lack of diverse chemical scaffolds extending into unexplored chemical and biological space. As a result, this has led to innovative reaction methodology and design strategies being used to deliver new NP-like scaffolds. In this thesis, access to four different NP-like scaffolds, by means of unique reaction methodology (Chapter I) or the pseudo-NP design principle (Chapter II) will be discussed.
A novel Rh(III)-catalysed C-H functionalisation reaction coupling N-Boc benzamides with ortho-substituted styrenes affording unique isoindolinone derivatives is disclosed in Chapter I. Experimental and computational investigations support that the high regioselectivity observed for these substrates results from the combination of the ortho-substituent on the styrene and the OBoc group of the benzamide. Furthermore, this scaffold was used to generate isoindolobenzazepine derivatives, via an additional SN2 reaction. Biological evaluation of selected compounds from both compound libraries revealed inhibitory activity in the Hedgehog (Hh)-dependent osteoblast differentiation of multipotent murine mesenchymal progenitor stem cells into osteoblasts. The pseudo-NP approach is a strategy involving the de-novo combination of NP fragments in novel arrangements with the dual aim of targeting novel chemical and biologically relevant space. A new class of pseudo-NPs combining indole- and tetrahydropyridine (THP)-fragments in a novel arrangement is described in Chapter II. The monopodal connectivity between the fragments, not yet observed in nature, was facilitated by a phosphine-catalysed asymmetric [4+2] annulation reaction, to obtain the pseudo-NP scaffold. Biological screening in various cell-based assays revealed the pseudo-NPs to be potent in reducing cellular kynurenine levels. Further investigation identified the mode-of-action to be the selective targeting and stabilisation of apo-indoleamine 2,3-dioxygenase 1 (apo-IDO1), a therapeutic target enzyme involved in immuno-suppression, linked to numerous disorders including cancer. Additionally, this pseudo-NP class was used to obtain the final NP-like class, in the form of bridged-bicyclic compounds. The synthesis of the pseudo-NP class validated the design principle with regard to unexplored fragment arrangements providing a route to compound classes endowed with unexpected or new bioactivity.2022-01-01T00:00:00ZProgrammable protein-DNA crosslinking for the direct capture and quantification of 5-formylcytosine
http://hdl.handle.net/2003/40995
Title: Programmable protein-DNA crosslinking for the direct capture and quantification of 5-formylcytosine
Authors: Gieß, Mario; Munoz-Lopez, Alvaro; Buchmuller, Benjamin; Kubik, Grzegorz; Summerer, Daniel
Abstract: 5-Formylcytosine (5fC) is an epigenetic nucleobase
of mammalian genomes that occurs as intermediate
of active DNA demethylation. 5fC uniquely interacts
and reacts with key nuclear proteins, indicating functions
in genome regulation. Transcription-activator-like effectors
(TALEs) are repeat-based DNA binding proteins that
can serve as probes for the direct, programmable recognition
and analysis of epigenetic nucleobases. However, no
TALE repeats for the selective recognition of 5fC are available,
and the typically low genomic levels of 5fC represent
a particular sensitivity challenge. We here advance TALEbased
nucleobase targeting from recognition to covalent
crosslinking. We report TALE repeats bearing the ketoneamino
acid p-acetylphenylalanine (pAcF) that universally
bind all mammalian cytosine nucleobases, but selectively
form diaminooxy-linker-mediated dioxime crosslinks to
5fC. We identify repeat-linker combinations enabling single
CpG resolution, and demonstrate the direct quantification
of 5fC levels in a human genome background by covalent
enrichment. This strategy provides a new avenue to
expand the application scope of programmable probes
with selectivity beyond A, G, T and C for epigenetic studies.2019-06-04T00:00:00ZEpigenetic chromatin modification by amber suppression technology
http://hdl.handle.net/2003/40994
Title: Epigenetic chromatin modification by amber suppression technology
Authors: Neumann, Heinz; Neumann-Staubitz, Petra; Witte, Anna; Summerer, Daniel
Abstract: The genetic incorporation of unnatural amino acids (UAAs) into proteins by amber suppression
technology provides unique avenues to study protein structure, function and interactions both in vitro
and in living cells and organisms. This approach has been particularly useful for studying mechanisms
of epigenetic chromatin regulation, since these extensively involve dynamic changes in structure,
complex formation and posttranslational modifications that are difficult to access by traditional
approaches. Here, we review recent achievements in this field, emphasizing UAAs that help to unravel
protein-protein interactions in cells by photo-crosslinking or that allow the biosynthesis of proteins
with defined posttranslational modifications for studying their function and turnover in vitro and in
cells.2018-02-22T00:00:00ZLight-activatable TET-dioxygenases reveal dynamics of 5-Methylcytosine oxidation and transcriptome reorganization
http://hdl.handle.net/2003/40993
Title: Light-activatable TET-dioxygenases reveal dynamics of 5-Methylcytosine oxidation and transcriptome reorganization
Authors: Palei, Shubhendu; Buchmuller, Benjamin; Wolffgramm, Jan; Muñoz-Lopez, Álvaro; Jung, Sascha; Czodrowski, Paul; Summerer, Daniel
Abstract: Ten-eleven-translocation (TET) dioxygenases
catalyze the oxidation of 5-methylcytosine (5mC), the central
epigenetic regulator of mammalian DNA. This activity dy-
namically reshapes epigenome and transcriptome by deposit-
ing oxidized 5mC derivatives, and initiating active DNA de-
methylation. However, studying this dynamic is hampered by
the inability to selectively activate individual TETs with tem-
poral control in cells. We report activation of TETs in mam-
malian cells by incorporation of genetically encoded 4,5-
dimethoxy-2-nitrobenzyl-L-serine as transient active site
block, and its subsequent deprotection with light. Our ap-
proach enables precise insights into the impact of cancer-
associated TET2 mutations on the kinetics of TET2 catalysis
in vivo, and allows time-resolved monitoring of target gene
activation and transcriptome reorganization. This sets a basis
for dissecting the order and kinetics of chromatin-associated
events triggered by TET catalysis, ranging from DNA de-
methylation to chromatin and transcription regulation.2020-04-14T00:00:00ZIdentification and characterization of inhibitors of hedgehog-induced osteoblast differentiation
http://hdl.handle.net/2003/40988
Title: Identification and characterization of inhibitors of hedgehog-induced osteoblast differentiation
Authors: Flegel, Jana Erika
Abstract: Hedgehog (Hh) signaling is one of the major pathways that is essentially required during embryonic development in vertebrates. Dysregulation during embryogenesis has been linked to severe developmental malformations including cyclopia and holoprosencephaly. Moreover, aberrant Hh signaling has been associated with several types of cancer, such as medulloblastoma and basal cell carcinoma. Over the past 40 years, studies have provided many insights into the current understanding of the Hh signaling pathway. Due promising potential for therapeutic modulation of this pathway, identification of small molecule modulators of Hh signaling is in high demand. Throughout this thesis, six compounds were identified as potent inhibitors of Hh-dependent osteogenesis during a phenotypic screening. Five of these compounds inhibited GLI2/3-dependent reporter activity and expression of Hh target genes and were thus classified as novel Hh pathway inhibitors. While four compounds, a Furo[3,2-b]pyridine, quinoline, pyrroloquinoline and 20-membered macrocycle derivative bind to the key signaling molecule smoothened (SMO), one compound, a 4-arylisoquinolone does not bind to the heptahelical bundle of this protein. An 8-oxotetrahydroprotoberberine derivate, termed Picoberin, inhibited Hh-dependent osteogenesis with a single-digit picomolar IC50 but did not modulate canonical Hh signaling. Despite its remarkable bioactivity, this small molecule was not cytotoxic at high concentrations in several cell lines. Global transcriptome and proteome profiling revealed that Picoberin activates Aryl hydrocarbon Receptor (AhR) signaling and led to the identification of AhR as the potential protein target. Subsequent functional evaluation of the influence of Picoberin on AhR signaling in several cell lines confirmed this target hypothesis. Moreover, chemical validations and genetic AhR depletion linked Picoberin mediated activation of AhR signaling to inhibition of Hh-dependent osteogenesis. Additionally, results obtained in this thesis provide evidence for crosstalk of AhR and Hh signaling and a functional role for AhR during osteoblast differentiation. AhR regulates xenobiotic metabolism as well as numerous physiological processes such as immune cell function, stem cell maintenance, and differentiation. Dysregulation of this ligand-activated transcription factor is observed in several diseases, including cancer. Historically, AhR is linked to mainly toxic effects. However, recent approval of the AhR agonist Tapinarof for treatment of plaque psoriasis and several studies exploring AhR in cancer provide evidence for potential therapeutic applications of AhR modulators. Picoberin could not only serve as a tool compound for AhR research but may potentially also provide a starting point for the development of further therapeutic AhR agonists.2022-01-01T00:00:00ZRedox-coupling with RPTPɣ regulates the growth factor response of EGFR
http://hdl.handle.net/2003/40974
Title: Redox-coupling with RPTPɣ regulates the growth factor response of EGFR
Authors: Joshi, Maitreyi S.
Abstract: Somatic cells rely on surface receptors like receptor tyrosine kinases (RTK) that are situated at the cell-surrounding interface to sense the environmental cues encoding the information about nutrients, cell growth and tissue damage. Proto-oncogenic epidermal growth factor receptor (EGFR) is a prominent RTK that binds to extracellular epidermal growth factor (EGF) and transduces the associated signal patterns inside the cell. Spatial re-organization of EGFR and its interactions with protein tyrosine phosphatases (PTPs) bridge EGF-binding to EGFR signaling to yield diverse cell fate outcomes. By exerting their phosphatase activities PTPs regulate the sensitivity and response dynamics of EGFR to growth factors. This raises a question on how do PTPs inhibit the spurious activation of EGFR without hindering its ligand dependent activation.
In this thesis, the coupling of EGFR with plasma membrane (PM)-based receptor-like protein tyrosine phosphatase-ɣ(RPTPɣ) and endoplasmic reticulum (ER)-bound T-cell protein tyrosine phosphatase (TCPTP) is examined to decipher the regulatory mechanisms that make EGFR capable of responding to the growth factor signals. We prove that expression of RPTPy is essential to suppress the anomalous activation of EGFR. The lack of RPTPɣ associated phosphatase activity makes the system susceptible to ligand-independent activation that turns it blind to the upcoming EGF signals. Consequently, cancer cells lacking RPTPɣ expression exhibit constitutive activity of EGFR. On the other hand, inhibition of RPTPɣ activity is necessary to trigger the activation of EGFR. This is achieved by EGF induced oxidation of the catalytic cysteine of RPTPɣ by the activation of reactive oxygen species (ROS) generating NOX-complexes. In the absence of NOX-activity, activation of EGFR is inhibited as a consequence of unhindered RPTPɣ catalytic activity. The regulation of EGFR response by the phosphatase activity of RPTPɣ and the EGFR kinase activity dependent regulation of RPTPɣ generates a ROS actuated toggle switch at the PM.
By designing a live-cell imaging probe for the spatial resolution of oxidized proteins, we show that the EGFR activity dependent oxidation of RPTPɣ happens at the PM by localized ROS production. Vesicular recycling reduces RPTPɣ and restores its catalytic activity by exposing it to the reducing environment of the cytoplasm. Additionally, endocytosis of EGFR establishes its interaction with TCPTP that regulates EGFR signaling duration by dephosphorylation. Therefore, vesicular recycling couples the redox cycle of RPTPɣ to the activity cycle of EGFR and also maintains the reversibility in the system. The interaction between RPTPɣ and monomeric EGFR spanning from the plasma membrane to recycling endosomes adds a spatial dimension to their regulation. By demonstrating its coupling with EGF-induced EGFR activity, we prove redox regulation of RPTPy as a major coordination mechanism that prevents EGFR from attaining uncontrolled signaling but allows for its EGF dependent signal propagation.2021-01-01T00:00:00ZThe role of innate lymphoid cells 1 and natural killer cells during drug induced liver damage
http://hdl.handle.net/2003/40955
Title: The role of innate lymphoid cells 1 and natural killer cells during drug induced liver damage
Authors: Metzler, Sarah
Abstract: Worldwide, approximately two million deaths per year are caused by different liver diseases. The understanding of the underlying mechanisms causing liver diseases remain incomplete. In the last two decades, the involvement of different immune cells during liver disease progression, regeneration and homeostasis have been closely investigated and offered new therapeutic strategies and targets.
Recently, natural killer (NK) cells have been observed to contribute to the progression of drug induced liver injury (DILI). In vitro results of primary human hepatocytes (PHH) and human hepatocyte cell lines pretreated with valproic acid, ketoconazole, promethazine and isoniazid showed enhanced expression of activating NK cell ligands. Moreover, enhanced NK cell activity was observed which was characterized by the higher interferon gamma (IFN) production and increased cytotoxicity against the pretreated hepatocytes. In order to validate the in vivo relevance of these findings and investigate the DILI mechanism, the first aim was to reproduce the enhanced NK cell activity ligand expression in drug pretreated primary mouse hepatocytes (PMH). The data of drug pretreated PMH revealed higher expression levels of activating NK cell ligands which were more pronounced in combination with tumor necrosis factor alpha (TNF). In the next step, the aim was to establish a drug induced liver injury mouse model with the most promising drug valproic acid. The attempts to establish a valproic acid induced liver injury mouse model were not successful, since the treatments did not cause increased NK cell numbers in the liver, higher expression of activating NK cell ligands on hepatocytes nor liver damage.
During the last decade, another immune cell type was identified which seem to be involved in liver damage, regenaration and homeostasis: innate lymphoid cell 1 (ILC1). Until today, there is not much known about ILC1s, however they have been described to possess a memory potential. Therefore, another aim of this thesis was to identify and characterize the memory potential of ILC1s after multiple applications of the hepatotoxic compound carbon tetrachloride (CCl4). After multiple doses of CCl4 injected in a 30 days interval, memory like ILC1s were discovered which were able to produce more interferon gamma (IFN). Further experiments revealed a transient effect of ILC1s in the liver, since higher cell numbers were observed on day one but until day three, the ILC1 cell numbers were again comparable to control cell numbers. Additionally, the memory effect of ILC1s is long-lasting. Nevertheless, the localization and function of memory like ILC1s in the liver need to be further investigated to evaluate the possible contribution to liver disease progression, regeneration and homeostasis.2022-01-01T00:00:00ZDiscovery and characterization of Pun1p, a putative component of the yeast fertilization synapse
http://hdl.handle.net/2003/40894
Title: Discovery and characterization of Pun1p, a putative component of the yeast fertilization synapse
Authors: Mainye, Sheila Kemunto
Abstract: Cell-cell fusion is a fundamental process in sexual reproduction and development but the mechanisms mediating and regulating this process are only now beginning to be uncovered. Despite recent progress in identifying key players in the plant and protist kingdoms, large gaps remain in other Eukaryotic lineages, including fungi. The cell fusion in the mating of the yeast Saccharomyces cerevisiae therefore presents itself as an ideal system to understand the molecular mechanisms underlying cell fusion in fungi. In an effort to identify novel components of the yeast fusion machinery and associated regulators, this thesis reports on the discovery and characterization of a membrane protein that negatively regulates the fusion of the plasma membrane (PM) which resembles tight junction claudins found in mammals.
A proteomics analysis of pheromone-regulated membrane proteins revealed that components of the MCC/eisosomes, a specialized furrow-like, membrane domain important for PM organization, were preferentially down-regulated during the pheromone response. An exception was Pun1p, a four-pass membrane-spanning protein with a conserved claudin motif, which was up-regulated in response to pheromone and was targeted to the transient junction established between two mating cells. Whereas deletion of PUN1 and its paralogs presented mild fusion defects, its function was distinctly revealed in a sensitized prm1Δ null background, where high-copy expression of PUN1 resulted in a partial inhibition of cell fusion. Further examination of the Pun1p inhibition activity in prm1Δ mutants revealed that PUN1 expression had no effect on lysis or formation of cytoplasmic bubbles, two prm1Δ phenotypes that occur after the cell wall (CW) has been remodeled. Instead, PUN1 expression inhibited fusion by enhancing the formation of a flat PM interface between two cells. However, contrary to previous reports, further analysis revealed that in a proportion of these mating pairs, the flat PM interface was structurally stabilized without any underlying CW material, suggesting the arrest occurred at a stage after CW remodeling via an unknown mechanism. Notably, the Pun1p inhibition activity was dosage and concentration-dependent and was enhanced with increasing concentration of Pun1p molecules across the mating junction, indicating additive functional trans-interactions. Analysis of the mechanistic basis of Pun1p activity revealed a claudin-like activity as a Pun1p mutant version of the conserved claudin motif lost its inhibitory activity. Additionally, the mutant protein exhibited localization defects and was modified at the monomeric level. Together, these results suggest that Pun1p is expressed in mating conditions and localized at the mating junction where it interacts with other unknown proteins to promote the formation of a PM junction-like structure similar to mammalian claudin-mediated tight junctions. The PM junction-like structure would function as a fusion fidelity checkpoint that negatively regulates PM fusion in the event of a compromised fusion machinery.
Finally, the identification of Pun1p as a novel component of the fusion machinery prompted further characterization of the mating junction by employing a Horseradish Peroxidase (HRP)-based proximity labeling approach. Using a HRP-Fus1p recombinant protein as a proof of principle, specific HRP-mediated labeling was observed at the shmoo tip of polarized cells, consistent with the pheromone-dependent localization of Fus1p. Although the ultimate objective of characterizing the labelled proteins was not plausible during the course of this thesis, these initial findings demonstrate the feasibility of this approach in yeast studies and its applicability in elucidating the molecular architecture of other similar junctions, a structure which we here refer to as a fertilization synapse.2021-01-01T00:00:00ZStrukturbiologische und zelluläre Charakterisierung Isoform-selektiver kovalent-allosterischer Akt-Inhibitoren
http://hdl.handle.net/2003/40893
Title: Strukturbiologische und zelluläre Charakterisierung Isoform-selektiver kovalent-allosterischer Akt-Inhibitoren
Authors: Depta, Laura
Abstract: Die Proteinkinase Akt, die aus drei Isoformen (Akt1/Akt2/Akt3) besteht, ist ein zentraler Knotenpunkt innerhalb des PI3K/Akt/mTOR-Signalwegs. Genomische Veränderungen wie aktivierende Mutationen in PI3K und Amplifikationen von AKT-Genen können eine Überaktivierung der Akt-Isoformen und damit einhergehend verschiedene Krankheiten auslösen. Trotz intensiver Forschung in den letzten Jahrzehnten sind mehrere klinische Studien mit potenziellen Arzneimittelkandidaten gescheitert. Verantwortlich dafür sind u. a. der Mangel an Informationen über Isoform-spezifische Funktionen im Zusammenhang mit menschlichen Krankheiten sowie die nicht selektive Adressierung der Isoformen und die damit verbundenen Nebenwirkungen. In dieser Arbeit war das Hauptziel, strukturbiologische und zelluläre Systeme zu etablieren, die die Grundlage für ein strukturbasiertes Wirkstoffdesign von Isoform-selektiven kovalent-allosterischen Akt-Inhibitoren (CAAIs) bilden. Zu diesem Zweck wurde zunächst ein detaillierter Sequenz- und Strukturvergleich der Akt-Isoformen durchgeführt. Die Strukturanalyse und ein fundiertes Konstruktdesign ermöglichten die Entwicklung effizienter Expressions- und Reinigungsstrategien. Weiterhin konnte im Rahmen von Kristallisationsstudien ein einzigartiger Einblick in die Akt2-Bindungstasche gewonnen werden, indem diese in Akt1 nachgebildet wurde. Dieser Einblick, kombiniert mit biochemischen und massenspektrometrischen Daten, ermöglichte eine Evaluierung einer fokussierten Substanzbibliothek und eine Verifizierung des von einem Homologiemodell abgeleiteten CAAI-Designansatzes. Um weiterführende zelluläre Studien zu ermöglichen, wurde ein Ba/F3-Akt-Isoform-abhängiges Modellsystem etabliert. Mit Hilfe dieses Modellsystems konnten biochemische Selektivitätsprofile der fokussierten Substanzbibliothek erstmalig in den zellulären Kontext übertragen werden und dienten als Grundlage für weitere Studien an der menschlichen Krebszelllinie PANC1. Darueber hinaus zeigten zellulären Studien an Akt-knock-out-Modellen die Vorteile der Isoform-selektiven CAAIs gegenüber invasiven genetischen knock-outs für die Entschlüsselung der Isoform-spezifischen Funktionen von Akt.; The protein kinase Akt, comprising three isoforms (Akt1/Akt2/Akt3), is a central signaling node within the PI3K/Akt/mTOR pathway. Genomic alterations such as activating mutations in PI3K and amplifications of AKT genes can trigger overactivation of Akt isoforms and concomitant various diseases. Despite intensive research in the last decades, several clinical trials with potential drug candidates have failed. Responsible for this are, among others, the lack of information on isoform-specific functions in the context of human disease and the non-selective targeting of the isoforms and associated side effects. In this work, the main objective was to establish structural biology and cellular systems that form the basis for structure-based drug design of isoform-selective covalent-allosteric Akt inhibitors (CAAIs). For this purpose, a detailed sequence and structural comparison of Akt isoforms was performed. Structural analysis and a founded construct design enabled the development of efficient expression and purification strategies. Furthermore, crystallization studies provided unique insight into the Akt2 binding pocket by mimicking it in Akt1. This insight, combined with biochemical and mass spectrometry data, allowed evaluation of a focused compound library and verification of the homology model-derived CAAI design approach. To enable further cellular studies, a Ba/F3-Akt isoform-dependent model system was established. Using this model system, biochemical selectivity profiles of the focused compound library could be transferred to the cellular context for the first time and served as a basis for further studies in the human cancer cell line PANC1. Furthermore, cellular studies on Akt knock-out models demonstrated the advantages of isoform-selective CAAIs over invasive genetic knock-outs for deciphering the isoform-specific functions of Akt.2022-01-01T00:00:00ZScanning protein surfaces with DNA-encoded libraries
http://hdl.handle.net/2003/40885
Title: Scanning protein surfaces with DNA-encoded libraries
Authors: Kunig, Verena B. K.; Potowski, Marco; Klika Škopić, Mateja; Brunschweiger, Andreas
Abstract: Understanding the ligandability of a target protein, defined as the capability of a protein to bind drug-like compounds on any site, can give important stimuli to drug-development projects. For instance, inhibition of protein–protein interactions usually depends on the identification of protein surface binders. DNA-encoded chemical libraries (DELs) allow scanning of protein surfaces with large chemical space. Encoded library selection screens uncovered several protein–protein interaction inhibitors and compounds binding to the surface of G protein-coupled receptors (GPCRs) and kinases. The protein surface-binding chemotypes from DELs are predominantly chemically modified and cyclized peptides, and functional small-molecule peptidomimetics. Peptoid libraries and structural peptidomimetics have been less studied in the DEL field, hinting at hitherto less populated chemical space and suggesting alternative library designs. Roughly a third of bioactive molecules evolved from smaller, target-focused libraries. They showcase the potential of encoded libraries to identify more potent molecules from weak, for example, fragment-like, starting points.2020-12-09T00:00:00ZMolecular working memory enables cells to navigate through changing complex environments
http://hdl.handle.net/2003/40863
Title: Molecular working memory enables cells to navigate through changing complex environments
Authors: Das, Abhishek
Abstract: In order to migrate within the complex environments of tissues and in organisms, cells
rely on sensing local chemical cues that are noisy, disrupted or change both in space and
time. The mechanism of how cells can maintain prolonged directional migration when
signals are irregular or conflicting, while being able to quickly re-adapt the directional
migration to novel sensory cues however has remained unknown. It has been predicted
that organization at criticality gives rise to a transient memory that is maintained
in the receptor’s activity via a slow-escaping remnant, a dynamical ”ghost” of the
attractor of polarized signaling state. Quantifying the dynamics of Epidermal Growth
Factor Receptor (EGFR) phosphorylation in live cells, as well as single-cell migration
assays in microfluidic devices, we provide experimental proof that the memory in cell
polarization results from dynamical trapping in a “ghost” state. We demonstrate that
this memory serves as a basis to integrate changing signals, but also to transform
the extracellular information for guidance of memory-directed migration. The results
therefore indicate that navigation in a complex environment is an emergent feature of
receptor networks organized at criticality. Generally, the molecular working memory
described here resembles the properties of working memory in neuronal networks, as
it enables information of previous signals to be temporally maintained while retaining
responsiveness to upcoming signals.; Um sich in der komplexen Umgebung von Geweben und anderen Organismen fortbewegen
zu können, sind Zellen auf die Wahrnehmung lokaler chemischer Signale angewiesen, die
verrauscht, unterbrochen, oder räumlich und zeitlich veränderlich sind. Ein Mechanismus
mit dem Zellen bei unregelmäßigen oder widersprüchlichen Signalen eine verlängerte,
gerichtete Fortbewegung beibehalten können, gleichzeitig aber in der Lage sind, die
Fortbewegung schnell an neue sensorische Signale anzupassen, ist derzeit noch unbekannt.
Es wurde vorhergesagt, dass die Organisation bei der Kritikalität zu einer transienten
Erinnerung eines polarisierten Signalzustandes der Aktivität eines Rezeptors führt, die
über einen langsam verschwindenden Rest, einen dynamischen „Geist“, des Attraktors
erhalten wird. Durch die Quantifizierung der Dynamik der Phosphorylierung des
epidermalen Wachstumsfaktor-Rezeptors (EGFR) in lebenden Zellen sowie durch
Einzelzell-Migrationsuntersuchungen in mikrofluidischen Geräten erbringen wir den
experimentellen Nachweis, dass das Gedächtnis in der Zellpolarisation aus dem
dynamischen Einfangen in einem "Geister“ Zustand resultiert. Wir demonstrieren, dass
dieses Gedächtnis als Basis für die Integration von veränderlichen Signalen dient, aber
auch die Transformation extrazellulärer Signale zur Lenkung der von der Gedächtnisdiktierten
Fortbewegung erlaubt. Die Ergebnisse deuten darauf hin, dass die Navigation
in einer komplexen Umgebung ein Merkmal ist, das aufgrund der Organisation des
Rezeptor Netzwerkes bei der Kritikalität entsteht. In den Grundzügen ähnelt das hier
beschriebene molekulare Arbeitsgedächtnis den Eigenschaften eines Arbeitsgedächtnisses
in neuronalen Netzwerken insofern, dass es die temporäre Erhaltung vorheriger Signale
ermöglicht, gleichzeitig aber die Reaktionsfähigkeit zu neuen Signalen erhält.2022-01-01T00:00:00ZLight-activation of DNA-methyltransferases
http://hdl.handle.net/2003/40846
Title: Light-activation of DNA-methyltransferases
Authors: Wolffgramm, Jan; Buchmuller, Benjamin; Palei, Shubhendu; Muñoz-López, Álvaro; Kanne, Julian; Janning, Petra; Schweiger, Michal R.; Summerer, Daniel
Abstract: 5-Methylcytosine (5mC), the central epigenetic mark of mammalian DNA, plays fundamental roles in chromatin regulation. 5mC is written onto genomes by DNA methyltransferases (DNMT), and perturbation of this process is an early event in carcinogenesis. However, studying 5mC functions is limited by the inability to control individual DNMTs with spatiotemporal resolution in vivo. We report light-control of DNMT catalysis by genetically encoding a photocaged cysteine as a catalytic residue. This enables translation of inactive DNMTs, their rapid activation by light-decaging, and subsequent monitoring of de novo DNA methylation. We provide insights into how cancer-related DNMT mutations alter de novo methylation in vivo, and demonstrate local and tuneable cytosine methylation by light-controlled DNMTs fused to a programmable transcription activator-like effector domain targeting pericentromeric satellite-3 DNA. We further study early events of transcriptome alterations upon DNMT-catalyzed cytosine methylation. Our study sets a basis to dissect the order and kinetics of diverse chromatin-associated events triggered by normal and aberrant DNA methylation.2021-04-07T00:00:00ZTowards DNA-encoded micellar chemistry: DNA-micelle association and environment sensitivity of catalysis
http://hdl.handle.net/2003/40838
Title: Towards DNA-encoded micellar chemistry: DNA-micelle association and environment sensitivity of catalysis
Authors: Klika Škopić, Mateja; Gramse, Christian; Oliva, Rosario; Pospich, Sabrina; Neukirch, Laura; Manisegaran, Magiliny; Raunser, Stefan; Winter, Roland; Weberskirch, Ralf; Brunschweiger, Andreas
Abstract: The development of DNA-compatible reaction methodologies is a central theme to advance DNA-encoded screening library technology. Recently, we were able to show that sulfonic acid-functionalized block copolymer micelles facilitated Brønsted acid-promoted reactions such as the Povarov reaction on DNA-coupled starting materials with minimal DNA degradation. Here, the impact of polymer composition on micelle shape, and reaction conversion was investigated. A dozen sulfonic acid-functionalized block copolymers of different molar mass and composition were prepared by RAFT polymerization and were tested in the Povarov reaction, removal of the Boc protective group, and the Biginelli reaction. The results showed trends in the polymer structure-micellar catalytic activity relationship. For instance, micelles composed of block copolymers with shorter acrylate ester chains formed smaller particles and tended to provide faster reaction kinetics. Moreover, fluorescence quenching experiments as well as circular dichroism spectroscopy showed that DNA-oligomer-conjugates, although highly water-soluble, accumulated very effectively in the micellar compartments, which is a prerequisite for carrying out a DNA-encoded reaction in the presence of polymer micelles.2021-05-12T00:00:00ZChemically stabilized DNA barcodes for DNA-encoded chemistry
http://hdl.handle.net/2003/40822
Title: Chemically stabilized DNA barcodes for DNA-encoded chemistry
Authors: Potowski, Marco; Kunig, Verena B. K.; Eberlein, Lukas; Vakalopoulos, Alexandros; Kast, Stefan M.; Brunschweiger, Andreas
Abstract: DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through structurally diverse molecules. Yet, the chemical reactivity of native DNA barcodes limits the toolbox of reactions for library design. Substituting the chemically vulnerable purines by 7-deazaadenine, which exhibits tautomerization stability similar to natural adenine with respect to the formation of stable Watson–Crick pairs, yielded ligation-competent, amplifiable, and readable DNA barcodes for encoded chemistry with enhanced stability against protic acid- and metal ion-promoted depurination. The barcode stability allowed for straightforward translation of 16 exemplary reactions that included isocyanide multicomponent reactions, acid-promoted Pictet–Spengler and Biginelli reactions, and metal-promoted pyrazole syntheses on controlled pore glass-coupled barcodes for diverse DEL design. The Boc protective group of reaction products offered a convenient handle for encoded compound purification.2021-06-21T00:00:00ZEnhanced ribozyme-catalyzed recombination and oligonucleotide assembly in peptide-RNA condensates
http://hdl.handle.net/2003/40796
Title: Enhanced ribozyme-catalyzed recombination and oligonucleotide assembly in peptide-RNA condensates
Authors: Le Vay, Kristian; Song, Emilie Yeonwha; Ghosh, Basusree; Tang, T.-Y. Dora; Mutschler, Hannes
Abstract: The ability of RNA to catalyze RNA ligation is critical to its central role in many prebiotic model scenarios, in particular the copying of information during self-replication. Prebiotically plausible ribozymes formed from short oligonucleotides can catalyze reversible RNA cleavage and ligation reactions, but harsh conditions or unusual scenarios are often required to promote folding and drive the reaction equilibrium towards ligation. Here, we demonstrate that ribozyme activity is greatly enhanced by charge-mediated phase separation with poly-L-lysine, which shifts the reaction equilibrium from cleavage in solution to ligation in peptide-RNA coaggregates and coacervates. This compartmentalization enables robust isothermal RNA assembly over a broad range of conditions, which can be leveraged to assemble long and complex RNAs from short fragments under mild conditions in the absence of exogenous activation chemistry, bridging the gap between pools of short oligomers and functional RNAs.2021-09-27T00:00:00ZStrukturbasiertes Design, Synthese und Evaluierung von Inhibitoren krebsrelevanter EGFR-Mutationen
http://hdl.handle.net/2003/40785
Title: Strukturbasiertes Design, Synthese und Evaluierung von Inhibitoren krebsrelevanter EGFR-Mutationen
Authors: Grabe, Tobias
Abstract: Heutzutage ist Krebs hinter den Herz-Kreislauf-Erkrankungen die zweithäufigste Todesursache in der westlichen Welt. Im Jahr 2020 stellten Lungen- und Bronchialkarzinome mit 1,8 Millionen Todesfällen weltweit die tödlichste aller Krebserkrankungen dar. Hiervon macht der nicht-kleinzellige Lungenkrebs (NSCLC, non-small cell lung carcinoma) etwa 80% aller Fälle aus. Eine Behandlung mit zytotoxischer Chemotherapie erzielt lediglich eine bescheidene Steigerung der Überlebensdauer der Patienten, geht aber zugleich mit starken Nebenwirkungen einher, welche die Lebensqualität erheblich reduzieren. Die Limitationen der Chemotherapie-Behandlung erforderten somit die Entwicklung neuartiger Therapieformen. Die heutzutage eingesetzte Therapie von NSCLC mit Inhibitoren sogenannter Treiberläsionen stellt ein Paradebeispiel der Präzisionsmedizin dar. Hierbei sind Mutanten-selektive Inhibitoren des Epidermalen Wachstumsfaktor-Rezeptors EGFR wie Osimertinib hervorzuheben. Bei der Behandlung von NSCLC mangelt es jedoch weiterhin an selektiven Inhibitoren zur Behandlung von gewissen Aktivierungs- und Resistenzmutationen des EGFR. Im Rahmen dieser Arbeit wurden verschiedene medizinal-chemische Ansätze zur Adressierung der C797S-Resistenzmutation und der Exon20-Insertions Aktivierungsmutanten untersucht. Für beide Problematiken konnten hochpotente Inhibitoren mit einzigartigen Selektivitätsprofilen generiert werden, welche somit potentielle Wirkstoff-Vorläufer für die Behandlung bislang nicht adressierbarer Mutanten des EGFR darstellen.2021-01-01T00:00:00ZIdentification of small-molecule modulators that enhance the ability of the immune system to eliminate cancer cells
http://hdl.handle.net/2003/40758
Title: Identification of small-molecule modulators that enhance the ability of the immune system to eliminate cancer cells
Authors: Hennes, Elisabeth
Abstract: The ability of the immune system to fight cancer is long-established whereas an in-depth understanding of how cancer cells can escape from immunosurveillance has only emerged over the last 20 year. This led to the development of the first groundbreaking cancer immunotherapies. However, the variety of cancer cell escape mechanisms is still not entirely elucidated, e.g., how cancer cells establish their immunosuppressive tumor microenvironment (TME). Although key components of the TME have been identified, only a few could be established as drug targets for the development of novel small-molecule drugs.
To discover new mechanisms to modulator the immunosuppressive features of the TME, two chemical genetic approaches were developed in the course of this thesis. The TME harbors various tumor-derived suppressive factors that inhibit effector immune cells, like natural killer (NK) cells, from eliminating cancer cells. In order to prevent NK cell suppression within the TME and to identify proteins or pathways involved in NK cell inhibition, a phenotypic assay was developed that facilitated the investigation of a small molecule library. In addition, the kynurenine (Kyn) metabolic pathway and its rate limiting enzyme indolamine 2, 3-dioxygenase (IDO1) plays a key role in immunosuppression within the TME. To prevent IDO1 activity a new cell-based assay was established to screen for small-molecule modulators that inhibit the Kyn pathway. Thereby, iDeg-1 was identified, the first monovalent small molecule degrader of IDO1.2022-01-01T00:00:00ZLight-activation of DNA-methyltransferases
http://hdl.handle.net/2003/40728
Title: Light-activation of DNA-methyltransferases
Authors: Wolffgramm, Jan
Abstract: The most prominent DNA modification in mammalian cells is the 5-methylation of cytosine (5mC) in a CpG context and several severe diseases like cancer are connected to aberrant cytosine methylation. 5mC is a dynamic key epigenetic modification regulating chromatin states and therefore gene transcription, and it is highly important to get new insights into the regulation and effects of such epigenetic mark. It is necessary to be able to write 5mC at user-defined genomic loci at specific time points to analyze locus specific and time-resolved downstream effects. 5mC is written by DNA methyltransferases (DNMTs) and several approaches were made to control 5mC levels either on a global or targeted gene level. However, these approaches had low spatial and/or temporal control and resulted in off-target effects. For now, it was not possible to control the catalytic activity of DNMTs itself. This work reports the direct light-control of in vivo DNMT activity to overcome drawbacks of previous approaches. A photocaged cysteine is genetically encoded on DNMT3a to replace the Cys710 which is essential for the catalytic activity. This leads to the expression of DNMT3a proteins in an inactive state and simultaneous activation of them is achieved by light irradiation. This results in the cleavage of the caging group and the remaining of an unmodified and active DNMT3a. Since upstream processes like transfection and protein expression are uncoupled from the actual catalytic activity after light-activation, the kinetics of 5mC writing alone can be monitored. This tool is used to study the effects of several DNMT3a mutations connected to acute myeloid leukemia on the catalytic activity, providing new in vivo observations to dissect the role of these mutations. In addition, it is a great advantage to be able to write 5mC at user-defined genomic loci to specifically alter the chromatin state or to monitor protein interactions associated with 5mC at such loci. Here, locus-specificity is reached by fusing a recombinant DNMT protein with a programmable transcription activator like effector protein. Thereby, inactive DNMTs are recruited to the target locus and activated at a given time point which prevents off-target methylation. It is noteworthy that the final 5mC level is tunable by adjusting the light irradiation time. Also, time-resolved effects of DNA methylation on the transcriptome are reported, providing data on the fast consequence of DNA methylation on gene expression. In conclusion, the ability to control the activity of DNMTs with least structural change by just incorporating a single non-canonical amino acid and the rapid activation of such by light gives previous unreachable spatio-temporal control over DNA methylation in living cells. With this tool, the kinetics of downstream effects of 5mC can be monitored and new insights into the 5mC-related epigenetic network can be obtained2021-01-01T00:00:00ZSynthese, in vitro und in vivo Evaluierung molekularer Sonden, PROTACs und PET-Tracern zur Adressierung der Proteinkinase Akt
http://hdl.handle.net/2003/40710
Title: Synthese, in vitro und in vivo Evaluierung molekularer Sonden, PROTACs und PET-Tracern zur Adressierung der Proteinkinase Akt
Authors: Lindemann, Marius
Abstract: Tumorerkrankungen stellen weltweit die zweithäufigste Todesursache dar und aufgrund der demographischen Entwicklung nimmt die Anzahl der Neuerkrankungen und Todesfälle stetig zu. Zur Adressierung der Proteinkinase Akt, ein hochrelevantes Protein in der Tumorentstehung, wurde für die präklinische Entwicklung die Hochskalierung eines kovalent-allosterischen Akt Inhibitor (CAAI) durchgeführt. Hiermit konnten in ersten Xenograft-Modellen vielversprechende Ergebnisse erhalten werden. Zur Optimierung der PK-Eigenschaften dieses CAAI wurde der chemische Raum erweitert. Die kleine fokussierte Substanzbibliothek auf Basis eines monozyklischen Pyridins lieferte neue Einblicke in die SAR der CAAIs. Dies ermöglichte das Design und die Synthese eines Akt-spezifischen Radioliganden, welcher in ersten in vivo Tiermodellen ein stabiles radioaktives Signal induzierte und durch weitere Optimierung zur Entwicklung eines PET-Tracers beitragen kann. Zur genaueren Analyse der Tumorbiologie von Akt wurden molekulare Sonden auf Basis der CAAIs entwickelt und in vitro etabliert. Als neuer innovativer Adressierungsansatz konnten erfolgreich die ersten drei Akt-spezifischen allosterischen PROTACs dargestellt werden, die einen Akt1-Abbau in Panc1-Zellen induzierten.; Tumor diseases represent the second leading cause of death worldwide and due to demographic trends the number of new cases and deaths is steadily increasing. To address the protein kinase Akt, a highly relevant protein in tumorigenesis, upscaling of a covalent-allosteric Akt inhibitor (CAAI) was performed for preclinical development. Promising results were obtained with this in initial xenograft models. To optimize the PK properties of this CAAI, the chemical space was expanded. The small focused compound library based on a monocyclic pyridine provided new insights into the SAR of CAAIs. This enabled the design and synthesis of an Akt-specific radioligand, which induced a stable radioactive signal in initial in vivo animal models and may contribute to the development of a PET tracer through further optimization. For a more detailed analysis of Akt tumor biology, molecular probes based on CAAIs were developed and established in vitro. As a new innovative targeting approach, the first three Akt-specific allosteric PROTACs were successfully displayed, which induced Akt1 degradation in Panc1 cells.2022-01-01T00:00:00ZDevelopment of contractive synthesis of cyclobutanes from pyrrolidines
http://hdl.handle.net/2003/40708
Title: Development of contractive synthesis of cyclobutanes from pyrrolidines
Authors: Hui, Chun-Ngai
Abstract: Carbocycles are omnipresence in chemical pharmaceuticals, biologically active natural products and organic functional materials. Construction of structurally intriguing, highly functionalized small carbocycles with congested stereocenters remain to be an intricate task in organic chemistry. Consistent endeavor such as innovation of chemical methodology and development of synthetic tactics has been made to improve the synthetic efficiency to these complex structures. In particular, the synthesis of carbocycle through ring contraction, which complies with the concept of synthetic efficiency in modern organic chemistry, has been widely applied in organic synthesis of complex architectures. In this thesis, the unprecedented, stereospecific and contractive synthesis of multi-substituted cyclobutanes from corresponding pyrrolidines is discussed. The reaction mechanism is investigated and the reaction was applied to the synthesis of cytotoxic natural product piperarborenine B.2021-01-01T00:00:00ZTranscriptomics-based identification of teratogens in differentiating hiPSCs: A novel in vitro test system
http://hdl.handle.net/2003/40707
Title: Transcriptomics-based identification of teratogens in differentiating hiPSCs: A novel in vitro test system
Authors: Seidel, Florian
Abstract: The knowledge about the developmental toxicity potential of substances is a matter of high concern in toxicology and medicine, especially if pregnant women need to take potentially teratogenic drugs to treat severe infections or diseases. The regulatory risk assessment of such substances is, however, very difficult, costly and time-consuming and relies currently, due to a lack of appropriate alternatives, solely on animal studies. During the last decades, much effort was put into the development and establishment of trustworthy in vitro-based test systems for the risk assessment of teratogenic substances, but not even one was approved for this purpose yet. In this PhD project, a new attempt was undertaken to develop such a system. The here presented novel system is based on the UKN1 assay, in which pluripotent human embryonic stem cells were differentiated to neuroepithelial precursor cells, and made considerable innovations to it by utilizing human induced pluripotent stem cells (hiPSCs), whole-transcriptome analyses and a thoroughly chosen set of 39 teratogenic and non-teratogenic substances that each were applied at two on human in vivo data-based concentrations to differentiating hiPSCs in this “UKN1 6-day” assay. In contrast to the non teratogenic compounds, the teratogens induced either significant gene expression alterations or a high toxicity in almost all cases, so that this test system was able to predict substances with a high accuracy of 90 %, a robust sensitivity of 83 % and an outstanding specificity of 100 %, what makes it therefore to a very high performing in vitro test system. Even a variation of the system, the UKN1 one-day assay, which shortened the total time requirements of the assay and replaced the whole-transcriptome analyses by a four biomarker-based RT-qPCR, could predict the compounds with an accuracy, sensitivity and specificity of 77 %, 63 % and 100 %, respectively. In addition to the core set of 39 substances, the UKN1 6-day assay was used to determine the health risks of parabens which are widely used as preservatives in cosmetics. No significant gene alterations were found in differentiated, paraben-exposed hiPSCs, and the obtained negative in vitro results could now support the existing in vivo data of paraben-exposed animals in accordance with a read-across-approach for ethylparaben, so that no further animal experiments would be needed for the regulatory risk assessment of ethylparaben.
In the near future, steps for a further optimization of the test system will be investigated like the application of targeted instead of whole-transcriptome analyses and multiple concentrations instead of just two. Furthermore, the number of tested compounds shall be increased. Although there is still a long way to go and many hurdles to overcome until this or another test system will be approved for the regulatory risk assessment of teratogens, the UKN1 6-day test system can already provide a tool for an in vitro teratogenicity screening of compounds and aid authorities and companies to assess the health risks of potentially teratogenic substances.2021-01-01T00:00:00ZDevelopment of an in vitro test battery for a test system to predict human drug-induced liver injury
http://hdl.handle.net/2003/40706
Title: Development of an in vitro test battery for a test system to predict human drug-induced liver injury
Authors: Brecklinghaus, Tim
Abstract: Drug-induced liver injury (DILI) is a major concern due to its poor predictability. Recently, we have developed an in vitro/in silico test system for the prediction of human DILI in relation to oral doses and blood concentrations. Additionally, two indices, the toxicity separation index (TSI) and the toxicity estimation index (TEI) were introduced for the quantitative evaluation of a test system and its input parameters. In this PhD-thesis, I studied whether extending the in vitro test battery of the test system, so far consisting of a cytotoxicity test in primary human hepatocytes, by additional functional readouts would lead to improved performance and thus allow a more accurate prediction. In total, three different approaches that address putative DILI-relevant mechanisms were explored. In the first approach, the influence of a bile acid mix on the cytotoxicity of in total 18 compounds in cultivated primary human hepatocytes was investigated. In summary, increased and decreased susceptibility to both hepatotoxic and non-hepatotoxic substances was observed with the addition of bile acids, which did not improve the TSI (0.79 -> 0.77) nor the TEI (0.73 -> 0.69) compared to cytotoxicity without additional bile acids. In the second approach, an assay was developed and evaluated that measures the inhibition of bile acid export carriers in primary human hepatocytes. In total 36 compounds were tested with the transport inhibtion assay and the cytotoxicity assay. In conclusion, the assay is able to detect bile acid export carrier inhibition and integration into the in vitro test battery improved TSI (0.77 -> 0.89) and TEI (0.69 -> 0.83) compared to cytotoxicity. In a third approach, intracellular lipid accumulation in HepG2 cells was investigated for a total of 60 compounds. Addition of lipid droplet accumulation to cytotoxicity improved TSI (0.74 -> 0.80) and TEI (0.67 -> 0.81). In summary, three assays were developed for the in vitro test battery of a test system to predict drug-induced liver injury. Quantitative analysis revealed that two of the three assays lead to improved separation of hepatotoxic and non-hepatotoxic compounds, as well as improved estimation of in vivo relevant blood concentrations. These improvements allow more accurate prediction of DILI by the test system.2021-01-01T00:00:00ZSupplement to: Multi-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
http://hdl.handle.net/2003/40674
Title: Supplement to: Multi-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
Authors: Nell, Patrick2021-01-01T00:00:00ZMacro-Pyrroquidines
http://hdl.handle.net/2003/40645
Title: Macro-Pyrroquidines
Authors: Niggemeyer, Georg2021-01-01T00:00:00ZNovel transition metal-free C−H bond functionalization methods for biologically important heterocycles synthesis
http://hdl.handle.net/2003/40644
Title: Novel transition metal-free C−H bond functionalization methods for biologically important heterocycles synthesis
Authors: Puthanveedu, Mahesh
Abstract: The development of transition metal-free reaction methodologies for the direct functionalization
of C−H bonds is an attractive area of research. These reactions offer atom-economic methods for
the synthesis and functionalization of valuable organic molecules in the absence of precious metal
catalysts under mild conditions. Novel methodologies have been established for biologically
important nitrogen containing molecules (Figure 1). A regioselective cross-coupling method for
the benzylation and alkynylation of privileged heterocyclic scaffolds like quinolines, isoquinolines
and pyridines were evaluated under organocatalytic conditions. Oxidized N-heterocyclic
compounds were reacted with organosilanes in presence of a fluoride catalyst. This method offers
a highly selective route to access C1-benzylated isoquinolines, C2-benzylated quinolines and
pyridines. Additionally, a unique sigma bond metathesis strategy is explored to obtain
symmetrically disubstituted acetylenes containing privileged scaffolds (Chapter 3).
Electroorganic chemistry has witnessed a renewed interest in recent years because of its profound
advantages over other methods such as oxidants mediated oxidative coupling reactions. Many
oxidants used in the organic reactions are potentially hazardous and even toxic. The risk of
handling such oxidants in stoichiometric amounts is high. Alternatively, electricity itself can be
employed as sole oxidant to carry out many redox reactions. Combining the field of transition
metal-free C−H functionalization chemistry with the revived electroorganic chemistry offers
unique advantages. Inspired by this proposal, an electrochemical dehydrogenative amination
including both intramolecular and intermolecular variants have been established. Detailed
mechanism involving a nitrenium ion intermediate has been proposed revealing the possible
generation of nitrenium ion intermediates under electrochemical oxidative conditions for the first
time (Chapter 4).
Direct C−H bond functionalization methods frequently give access to the kind of molecules which
were never accessed before. These molecules however might be relevant in terms of biological
properties. Therefore, the evaluation of bioactivities for novel molecules synthesized by direct
C−H bond functionalization methods are highly significant. A novel physiological, morphogenic
cellular screening system that is focused on the bone morphogenetic pathway revealed chromones
as potential BMP activator chemotypes. The key step in the synthesis of these chromone
compounds is an oxidative C−H amination in presence of molecular iodine and base. The active hits were resynthesized in adequate amounts for in vitro and in vivo studies. Additionally, based
on the molecular structure of active hits, novel derivatives were designed and synthesized.
Afterwards, biological experiments were carried out in collaboration to understand the structure
activity relationship of various chromones as BMP effectors (Chapter 5).; Die Entwicklung von übergangsmetallfreien Reaktionsmethoden für die direkte
Funktionalisierung von C−H-Bindungen ist ein attraktives Forschungsgebiet. Diese Reaktionen
bieten verbesserte, atomökonomische Methoden für die Synthese und Funktionalisierung
wertvoller organischer Moleküle in Abwesenheit von Edelmetallkatalysatoren unter relativ milden
Bedingungen. In diesem Zusammenhang wurden neue Methoden für biologisch relevante
stickstoffhaltige Moleküle entwickelt (Abbildung 1). Zunächst wurde eine regioselektive
Kreuzkupplungsmethode für die Benzylierung und Alkynylierung privilegierter heterozyklischer
Strukturgerüste wie Chinoline, Isochinoline und Pyridine unter organokatalytischen Bedingungen
untersucht. Voroxidierte N-heterozyklische Verbindungen wurden mit Organosilanen in
Anwesenheit eines Fluoridaktivators umgesetzt. Diese Methode bietet einen hochselektiven
Zugang zu C1-benzylierten Isochinolinen, C2-benzylierten Chinolinen und Pyridinen. Zusätzlich
wird eine einzigartige Sigma-Bindungs-Metathesestrategie untersucht, um symmetrisch
disubstituierte Acetylene zu erhalten, die an beiden Enden privilegierte Gerüste aufweisen. Diese
Verbindungen wurden auch auf potenzielle Bioaktivität untersucht (Kapitel 3). Die
elektroorganische Chemie hat in den letzten Jahren aufgrund ihrer tiefgreifenden Vorteile
gegenüber anderen Methoden, wie z. B. durch chemische Oxidationsmittel vermittelte oxidative
Kupplungsreaktionen, ein neues Interesse geweckt. Viele verwendeten Oxidationsmittel in
organischen Reaktionen sind potenziell gefährlich und sogar giftig. Der Umgang mit solchen
Oxidationsmitteln in stöchiometrischen Mengen ist mit hohem Risiko verbunden. Alternativ kann
Elektrizität selbst als gezieltes Oxidationsmittel eingesetzt werden, um viele Redoxreaktionen
durchzuführen. Die Kombination von übergangsmetallfreien C−H-Funktionalisierungschemie mit
der wiederbelebten elektroorganischen Chemie bietet einzigartige Vorteile. Inspiriert von diesem
Ansatz wurde eine elektrochemische dehydrierende Aminierung entwickelt, die sowohl
intramolekulare als auch intermolekulare Varianten umfasst. Ein detaillierter Mechanismus, an
dem ein Nitrenium-Ionen-Zwischenprodukt beteiligt ist, welcher zum ersten Mal die mögliche
Bildung von Nitrenium-Ionen Zwischenprodukten unter elektrochemischen oxidativen
Bedingungen aufzeigt, wurde vorgeschlagen (Kapitel 4).
Direkte C−H-Bindungsfunktionalisierungsmethoden ermöglichen häufig den Zugang zu
Molekülen, die bisher nicht bzw. erschwert zugänglich waren. Diese Moleküle könnten jedoch im Hinblick auf biologische Eigenschaften von hoher Bedeutung sein. Daher ist die Untersuchung der
Bioaktivität neuartiger Moleküle, die mit Hilfe direkter C−H-Bindungsfunktionalisierungsmethoden synthetisiert wurden, von großer Bedeutung. Ein neuartiges physiologisches, morphogenetisches zelluläres Screening-System, das sich auf den Knochenmorphogenese-Weg (BMP-Weg) konzentriert, hat zum Beispiel Chromone als potenziellen BMP-Aktivator-Chemotyp enthüllt. Der Schlüsselschritt bei der Synthese dieser
Chromonverbindungen ist eine oxidative CH-Aminierung in Gegenwart von molekularem Jod und
einer Base. Die aktiven Verbindungen wurden in ausreichenden Mengen für In-vitro- und In-vivo-
Studien resynthetisiert. Darüber hinaus wurden auf der Grundlage der Molekularstruktur der
aktiven Moleküle neue Derivate entworfen und synthetisiert. Anschließend wurden in
Zusammenarbeit biologische Experimente durchgeführt, um die Struktur-Aktivitäts-Beziehung
der verschiedenen Chromone als BMP-Effektoren zu analysieren.2021-01-01T00:00:00ZDevelopment of metal-free methodologies for the synthesis of biologically relevant compounds
http://hdl.handle.net/2003/40643
Title: Development of metal-free methodologies for the synthesis of biologically relevant compounds
Authors: Polychronidou, Vasiliki
Abstract: The establishment of novel concepts for metal-free transformations in modern synthetic chemistry is in high demand. For decades, synthetic chemists were relying on transition metal catalysis to accomplish important classes of organic reactions. However, nowadays the need of society towards more sustainable and greener chemistry is higher than ever. Therefore, metal-free strategies can provide an environmentally benign and economical solution for the synthesis of simple building blocks to access “target molecules”.
In the present thesis, the development of novel metal free methodologies for the construction of biologically interesting nitrogen-containing molecules (chapter 3) as well as for asymmetric oxidative transformations (chapter 4) has been studied.
The modular synthesis of 1,6-dihydropyridine scaffolds has been investigated in a metal-free manner. To achieve this, a combination of vinyliminophospharenes and ketones in an aza-Wittig/6π-electrocyclization sequence was employed. The transformation proceeded via the generation of an azatriene, the key intermediate in the reaction sequence, which would further cyclize in a 6pi electron disrotatory mode. A wide variety of unprecedented 1,6-dihydropyridines with quaternary centers was obtained with yields up to 97%, due to the high functional group tolerance of the method. Further structural modifications on the 1,6-dihydropyridine scaffold through hydrogenation and Diels-Alder reaction allowed a facile access to the corresponding tetrahydropyridine, piperidine and isoquinuclidine moieties. Furthermore, the development of an asymmetric variant of this methodology was attempted using various chiral Brønsted acids which resulted in 1,6-dihydropyridines with poor enantioselectivities.
In the second part of the thesis, the reactivity and stereoselectivity of chiral hypervalent iodine(III) reagents in asymmetric oxidative transformations were explored. These compounds are well-known substitutes for transition metal-catalyzed reactions. Therefore, the rational design and rapid synthesis of a series of reported and novel C2-symmetric iodoarenes endowed with different chiral features was accomplished. To evaluate both the catalytic activity and the asymmetry-inducing ability of the synthesized iodoarenes, they were applied in enantioselective α-functionalization of carbonyl compounds and specifically in the α-tosyloxylation of propiophenone and ethyl 2-methylacetoacetate, and lactonization of 4-benzoylbutyric acid. Even though, they proved to be able to catalyze these oxidative transformations yielding the products in moderate to good yields, their ability to exert stereocontrol was inadequate.; Die Entwicklung von neuen Konzepten für metallfreie Umwandlungen in der modernen Synthese-Chemie ist sehr gefragt. Jahrzehntelang insistierten Synthesechemiker auf die Katalyse von Übergangsmetallen, um wichtige Klassen organischer Reaktionen durchzuführen. Heutzutage ist das Bedürfnis der Gesellschaft nach einer nachhaltigeren und umweltfreundlicheren Chemie jedoch größer denn je. Daher können metallfreie Strategien eine umweltfreundliche und wirtschaftliche Lösung für die Synthese einfacher Bausteine darstellen, um Zugang zu "Zielmolekülen" zu erhalten.
In der vorliegenden Arbeit wurde die Entwicklung neuartiger metallfreier Methoden für die Herstellung biologisch relevanter stickstoffhaltiger Moleküle (Kapitel 3) sowie für asymmetrische oxidative Transformationen (Kapitel 4) untersucht.
Die modulare Synthese von 1,6-Dihydropyridin-Gerüsten wurde über eine metallfreie Methode untersucht. Dazu wurden Vinyliminophospharene und Ketone in einer Aza-Wittig/6π-Elektrozyklisierungssequenz eingesetzt. Die Umwandlung verlief über die Bildung eines Aza-Triens, ein Schlüsselintermediat in der Reaktionssequenz, das über einen 6π-disrotatorischen Verlauf weiter zyklisiert. Aufgrund der hohen Toleranz von funktionellen Gruppen dieser Methode, wurde eine große Vielfalt an neuen 1,6- Dihydropyridinen mit quaternären Zentren mit einer Ausbeute von bis 97% erhalten. Weitere strukturelle Modifikationen am 1,6-Dihydropyridin-Gerüst durch Hydrierung und Diels-Alder-Reaktion ermöglichten einen einfachen Zugang zu den entsprechenden Tetrahydropyridinen, Piperidinen und Isochinolidinen. Schließlich wurde die Entwicklung einer asymmetrischen Variante dieser Methode unter Verwendung von verschiedenen chiralen Brønsted-Säuren versucht, die zu 1,6-Dihydropyridinen mit niedrigen Enantioselektivitäten führten.
Im zweiten Teil der Arbeit wurden die Reaktivität und Stereoselektivität von chiralen hypervalenten Iod(III)-Reagenzien in der asymmetrischen oxidativen Umwandlungen untersucht. Diese Verbindungen sind bekannte Alternativen für Übergangsmetall-katalysierte Reaktionen. Ein strukturelles Design sowie eine vereinfachte Synthese von neuartigen C2-symmetrischen Iodoarenen mit verschiedenen chiralen Eigenschaften wurde durchgeführt. Um sowohl die katalytische Aktivität als auch die Asymmetrie-induzierende Fähigkeit der synthetisierten Jodarene zu bewerten, wurden diese in einer enantioselektiven α-Funktionalisierung von
Carbonylverbindungen wie der α-Tosyloxylierung von Propiophenon und Ethyl-2-
Methylacetoacetat sowie der Lactonisierung von 4-Benzoylbuttersäure angewendet. Obwohl diese
sich als fähig erwiesen, die oxidativen Umwandlungen zur Produkt Bildung zu katalysieren, war
das Potential bezüglich der Stereokontrolle nicht ausreichend.2021-01-01T00:00:00ZCellular model system to dissect the isoform-selectivity of Akt inhibitors
http://hdl.handle.net/2003/40596
Title: Cellular model system to dissect the isoform-selectivity of Akt inhibitors
Authors: Quambusch, Lena; Depta, Laura; Landel, Ina; Lubeck, Melissa; Kirschner, Tonia; Nabert, Jonas; Uhlenbrock, Niklas; Weisner, Jörn; Kostka, Michael; Levy, Laura M.; Schultz-Fademrecht, Carsten; Glanemann, Franziska; Althoff, Kristina; Müller, Matthias P.; Siveke, Jens T.; Rauh, Daniel
Abstract: The protein kinase Akt plays a pivotal role in cellular processes. However, its isoforms’ distinct functions have not been resolved to date, mainly due to the lack of suitable biochemical and cellular tools. Against this background, we present the development of an isoform-dependent Ba/F3 model system to translate biochemical results on isoform specificity to the cellular level. Our cellular model system complemented by protein X-ray crystallography and structure-based ligand design results in covalent-allosteric Akt inhibitors with unique selectivity profiles. In a first proof-of-concept, the developed molecules allow studies on isoform-selective effects of Akt inhibition in cancer cells. Thus, this study will pave the way to resolve isoform-selective roles in health and disease and foster the development of next-generation therapeutics with superior on-target properties.2021-09-06T00:00:00ZMulti-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
http://hdl.handle.net/2003/40593
Title: Multi-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
Authors: Nell, Patrick
Abstract: Humane, induzierte pluripotente Stammzellen (iPSC) zeigen Potenzial für die therapeutische Applikation in der personalisierten Medizin, sowie die Verwendung zur Entwicklung von Modellsystemen in pharmakologischen und toxikologischen Studien. Die Differenzierung von iPSC zu Hepatozyten-ähnlichen Zellen wurde durch eine Vielzahl an Protokollen realisiert, wobei die resultierenden Zellen ein variierendes Ausmaß phänotypischer Reife aufweisen. Die genomweite Expression zeigt enorme Unterschiede zu primären Hepatozyten (PHH), veranschaulicht an Hand der Expression von Leber- wie auch Darm-assoziierten Genen. In dieser Arbeit wurde die Differenzierung von iPSC über definitive Endoderm (DE) zu HLC mittels Einzelzell- sowie Bulk-RNA-Sequenzierung und epigenetischen Analysen charakterisiert. Für die Auswertung wurde eine überwachte Strategie zur Gruppierung von Genen mit ähnlichen Expressionsverläufen in Differenzierungsmuster-Gruppen (DPG) entwickelt. Diese DPGs wurden mit bioinformatischen Methoden analysiert um Gen-Netzwerke und Regulatoren zu identifizieren, die zu gewünschter oder auch unerwünschter Differenzierung beitragen. Epigenetische Analysen konnten zeigen, dass eine Verbindung zwischen der Zugänglichkeit von Chromatin und der Expression erwünschter und unerwünschter Faktoren besteht und dass HLC im Vergleich zu PHH eine globale hyper-Methylierung ihrer Promoterregionen aufweisen. Dies legt grundsätzlich nahe, dass HLC nicht die für Hepatozyten charakteristische epigenetische Landschaft ausbilden. Außerdem konnte mittels Einzelzell-Sequenzierung gezeigt werden, dass HLC eine einzige Zellpopulation darstellen, welche in einem Hybridzustand verharrt, in dem Hepatozyten-assoziierte Gene zusammen mit unerwünschten – hauptsächlich Darm-assoziierten - Genen, die weder in adulten noch fetalen primären Hepatozyten vorkommen, exprimiert werden. Diese Ergebnisse verdeutlichen, dass in der Differenzierung von HLC in vitro wichtige Signale fehlen, die der Differenzierung von Hepatozyten in vivo zu Grunde liegen. Analysen zur Überrepräsentation von Gen-Regulatoren, die zwischen HLC, fetalen und adulten primären Hepatozyten unterschiedlich exprimiert werden, deuteten auf eine zentrale Rolle für den Kernrezeptor FXR hin. Die Kombination von FXR Expression und Aktivierung des Rezeptors durch Agonisten konnte die Expression von Hepatozyten-assoziierten Genen fördern, während die Expression unerwünschter Darmgene teils unterdrückt wurde. Auf diese Weise konnte gezeigt werden, wie die HLC Differenzierung durch gezielte Manipulation von Gennetzwerken verbessert werden kann.; Human induced pluripotent stem cell (iPSC)-derived cells hold much promise for future cell therapy applications in the field of personalized regenerative medicine and offer an unlimited source of cell material for all branches of life-science associated research, including their use as model systems in pharmacological and toxicological studies. Differentiation of iPSC to hepatocyte-like cells (HLC) has been achieved in vitro by a number of differentiation protocols showing varying degrees of phenotypic maturity. However, their genome-wide expression patterns still differ significantly from primary human hepatocytes (PHH) in that they express liver- as well as intestine-associated genes. In this PhD thesis, differentiation of iPSC via definitive endoderm (DE) to HLC was characterized by single cell, as well as bulk RNA-sequencing with complementary epigenetic analyses. A supervised clustering strategy was developed to identify genes that exhibit similar expression dynamics during the differentiation of iPSC to HLC and assign them into differentiation pattern groups (DPG). These DPGs were analyzed by bioinformatics procedures to identify gene networks and regulatory factors that are likely to contribute to hepatocyte (favorable) or non-hepatocyte (adverse) differentiation. Epigenetic analysis showed global hypermethylation of promoters in iPSC- derived cells compared to PHH and that there is a link between chromatin accessibility and favorable and adverse gene expression, suggesting that HLC fail to acquire an epigenetic landscape characteristic of PHH. Strikingly, single cell RNA-seq revealed that favorable and adverse gene expression occurs in a single population of HLC, rather than subpopulations. Thus, HLC exist in a hybrid state, where hepatocyte-associated genes are expressed in concert with genes that are not expressed in PHH or fetal hepatocytes (FH) - mostly intestinal genes - within the same cell. This finding highlights a substantial lack of developmental cues required to guide lineage-specific differentiation of iPSC to hepatocytes. Overrepresentation analysis at the bulk level, as well as regulon analysis at the single cell level, identified sets of regulatory factors that differ between HLC, FH, and PHH, hinting at a central role for the nuclear receptor FXR in the functional maturation of HLC. Combined FXR expression plus agonist exposure enhanced the expression of hepatocyte-associated genes and suppressed undesired non-liver gene expression, thereby improving HLC similarity to PHH.2021-01-01T00:00:00ZRNA degradation using small molecule-based recruiters of RNase L
http://hdl.handle.net/2003/40562
Title: RNA degradation using small molecule-based recruiters of RNase L
Authors: Stenbratt, Carl Leonard
Abstract: The human genome is mainly transcribed into non-coding RNAs that are not translated into proteins. With increasing understanding of function through continued research it has become evident that parts of the transcriptome are disease-related. RNAs are becoming increasingly important targets for the development of novel medicines using chemical modalities such as small molecules, bifunctional molecules, peptides, oligonucleotides and conjugates. Yet only part of what is possible to achieve by targeting non-coding RNAs has been discovered, which allows for the development of therapeutics with novel mode of actions beyond current drugs.
Described in this thesis are strategies targeting RNAs for catalytic degradation. The design, synthesis, characterization and biochemical evaluation of both 2-aminothiophene-containing heterocyclic molecules and thiophenones identified a suitable compound class for activation of RNase L. Development of the novel RITAC strategy was based on chemical conjugates, containing thiophenones, for targeted degradation of the RNA interactome of RNA-binding proteins via recruitment of RNase L. The RNA-binding protein WDR5 was used in a proof-of-concept study, where obtained heterobifunctional molecules, targeting an allosteric binding site within WDR5, may serve to identify new protein-RNA interactions and to develop therapeutics for the treatment of acute myeloid leukemia. Additionally, the scope of the RIBOTAC strategy was expanded using thienopyridines to recruit RNase L for degradation of the HIV-1 RNA genome by binding to the transactivation response element. Obtained heterobifunctional molecules may serve as therapeutics for the treatment of HIV-1 infections.2021-01-01T00:00:00ZThe small GTPase RAB18: Insights into cellular steatosis, lipophagy and the non-alcoholic fatty liver disease
http://hdl.handle.net/2003/40554
Title: The small GTPase RAB18: Insights into cellular steatosis, lipophagy and the non-alcoholic fatty liver disease
Authors: Rieck, Adrian
Abstract: Non-alcoholic fatty liver disease is a rapidly growing concern for public health. Its most prevalent marker is steatosis, which is the accumulation of large lipid storage organelles called lipid droplets (LD) in the hepatocytes. RAB18, a member of the Rab family, localizes to the LD membrane. Rab family proteins are regulators of cellular membrane trafficking, therefore RAB18 is expected to play a role regulating LD biology. The presented work aims to elucidate this role as well as the mechanisms behind the localization of RAB18 to the LD membrane.
The localization of RAB18 was investigated by overexpressing mutant RAB18 variants in HepG2 cells. RAB18 localization was observed to depend on the reversible cyclical palmitoylation of its C-terminus. Using FRAP experiments, it could be shown that targeting the palmitoylation machinery with small molecule inhibitors modulated RAB18 localization. This coincided with changes in LD size in cells treated with de-palmitoylation inhibitors.
An overall increase in LD size was observed in HepG2 cells with RAB18 downregulation. The wild type LD size in these cells was restored by the inhibition of autophagy. This size reduction was due to newly created LDs. Inhibition of autophagy prior to LD accumulation was subsequently tested in vitro on primary human hepatocytes in sandwich culture. Inhibition of autophagy by chloroquine resulted in a dose dependent rise in LD number and a decrease in average LD size in these cells.
These effects could be translated to the in vivo situation in mice. Daily chloroquine injection of mice on a steatogenic diet resulted in a significant decrease of LD size in vivo. Conversely, no changes were detected in the blood-values of treated mice compared with the control.
This thesis demonstrates, that RAB18 localizes to the LD via a C-terminal acylation cycle. RAB18 reduces the size of LDs by modulating autophagy of newly formed LDs. This mechanism is important for LD number and size regulation in hepatocytes.2021-01-01T00:00:00ZDeciphering strand-asymmetrically modified CpG dyads in the DNA double-helix
http://hdl.handle.net/2003/40553
Title: Deciphering strand-asymmetrically modified CpG dyads in the DNA double-helix
Authors: Buchmuller, Benjamin Christopher
Abstract: Alle Lebewesen müssen dafür Sorge tragen, in ihrem umfangreichen Erbgut die gerade für sie überlebensnotwendigen Gene von denen zu unterscheiden, die nicht gebraucht werden. Insbesondere mehrzellige Organismen müssen diesen Prozess zellspezifisch koordinieren, trotz dessen, dass hier dieselbe Erbinformation in allen Zellen des Individuums vorliegt. Ein Mechanismus, welcher diesem Zwecke dient, ist die Modifikation von DNA-Nukleobasen, den Bausteinen des Trägers der Erbinformation.
In Säugetieren wie Mensch und Maus kommt hierbei der Methylierung der DNA-Nukleobase Cytosin am Kohlenstoffatom C5 des Pyrimidinrings eine besondere Rolle zu. Sie findet auf beiden Strängen der DNA-Doppelhelix innerhalb des kurzen Sequenzpalindroms CpG statt und trägt entscheidend dazu bei, dass hier ortsspezifisch andere molekulare Interaktionen für die Expression der Erbinformationen notwendig werden. Da das Produkt 5-Methylcytosin für weitere enzymatische Modifikationen wie der Oxidation zu 5-Hydroxymethylcytosin, 5-Formylcytosin oder 5-Carboxycytosin zur Verfügung steht, können unterschiedliche Kombinationen dieser Cytosinderivate mit gänzlich einzigartigen chemischen Eigenschaften an den komplementären CpG-Paaren im DNA-Doppelstrang vorliegen. Ein Aspekt, der unter dem Gesichtspunkt der epigenetischen Funktion dieser Derivate in Ermangelung technologischer Innovation sie in natürlichem Chromatin zu untersuchen, bislang kaum erschlossen werden konnte. Inwiefern es nun möglich ist, solche Strang-symmetrischen oder Strang-asymmetrischen Kombinationen von Cytosinderivaten in diesen CpG-Paaren auf molekularer Ebene in der DNA-Doppelhelix zu erkennen und somit gegebenenfalls zu entschlüsseln, ist Gegenstand der vorliegenden Arbeit.
Ausgehend von verschiedenen Homologen einer Proteindomäne, welche symmetrisch methylierte CpG-Paare erkennen, den Methyl-CpG-bindenden Domänen (MBD), wurden aufgrund struktureller Erwägungen und funktionaler Studien der MBD–DNA-Binding, degenerierte Proteinvariantenbibliotheken erstellt. Mithilfe eines hierfür eigens entwickelten Hochdurchsatzverfahrens gelang es, Varianten zu identifizieren, die nahezu selektiv eine aus fünfzehn Paarungen obiger Cytosinderivate im DNA-Doppelstrang erkennen. Neben allgemeinen Substitutionsprofilen für verschiedene Paarungen wurden im Speziellen mehrere MBD-Varianten entdeckt, die eine neue, natürlicherweise nicht vorhandene Selektivität für 5-Hydroxymethyl- und 5-Carboxymethylcytosin-haltige CpG-Paarungen aufwiesen. Aus der weiteren biochemischen und strukturellen Charakterisierung der Bindespezifität konnten einige Erkenntnisse über die molekulare Erkennung Strang-asymmetrisch modifizierter CpG-Paarungen gewonnen werden, welche in Zukunft als Schlüssel dienen können, die epigentische Funktion der Cytosinmodifizierung im humanen Genom mithilfe solcher speziell auf sie zugeschnittenen Sonden zu entschlüsseln.2021-01-01T00:00:00ZDNA-kodierte Substanzbibliotheken: Chemische Stabilisierung der DNA, Entwicklung neuer Synthesemethoden und Identifizierung von TEAD-YAP-Inhibitoren
http://hdl.handle.net/2003/40530
Title: DNA-kodierte Substanzbibliotheken: Chemische Stabilisierung der DNA, Entwicklung neuer Synthesemethoden und Identifizierung von TEAD-YAP-Inhibitoren
Authors: Kunig, Verena B. K.
Abstract: Die Technologie der DNA-kodierten Substanzbibliotheken (DELs) hat sich in den letzten Jahren als eine vielversprechende Alternative zum Hochdurchsatz-Screening zur Identifizierung kleiner organischer Moleküle, welche mit pharmazeutisch relevanten, biologischen Zielstrukturen interagieren, etabliert. DELs bestehen aus einer Vielzahl an DNA-kodierten Molekülen, welche gleichzeitig in Affinitäts-basierten Selektionsassays gegenüber einer Zielstruktur getestet und im Anschluss anhand ihrer einzigartigen DNA-Sequenz leicht „entschlüsselt“ werden können. Ein Großteil der in der Literatur beschriebenen DELs wird über „split and pool“-Synthesen in wässriger Lösung synthetisiert. Dies hat zur Folge, dass viele gängige Synthesemethoden der organischen Chemie, die auf trockene Lösungsmittel angewiesen sind, nicht für die DEL-Synthese zur Anwendung kommen können. Eine weitere Limitierung zur Herstellung von DELs stellt die Stabilität der DNA gegenüber verschiedenen Reaktionsbedingungen dar. Viele als Katalysatoren standardmäßig in der präparativen organischen Chemie eingesetzte Metallsalze sowie stark saure Reaktionsbedingungen können in der DEL-Synthese nicht verwendet werden.
Im Rahmen dieser Arbeit wurde der Fokus auf die Synthese von DELs basierend auf einer Festphasenstrategie gelegt. Diese bietet neben der freien Wahl des Lösungsmittels den Vorteil, dass die Nukleobasen der DNA vollständig geschützt vorliegen und somit die gesamte DNA eine höhere Stabilität aufweist. Im ersten Teil der Arbeit wird die Synthese einer Indol-fokussierten DNA-kodierten Substanzbibliothek, ausgehend von dem chemisch sehr stabilen, controlled pore glass (CPG)-gebundenen Hexathymidin („hexT“)-Adapteroligonukleotid beschrieben (thymidine-initiated DNA-encoded chemistry, TIDEC). Die im Vergleich zu anderen DELs recht kleine Bibliothek von 8.112 Molekülen konnte im Selektionsscreening vielversprechende Wirkstoffkandidaten für schwierig zu adressierende Proteine wie dem Transkriptionsfaktor TEAD4 liefern. Das in diesem Verfahren verwendete Adapteroligonukleotid hexT erlaubt jedoch keine Kodierung von Startmaterialien. Diese Einschränkung wird im weiteren Verlauf der Arbeit adressiert durch die Überführung weiterer organischer Synthesemethoden (Ugi-Vierkomponentenreaktion, Ugi-Azid-Vierkomponentenreaktion, Ugi-Vierkomponenten-aza-Wittig-Reaktion, Groebke-Blackburn-Bienaymé-Dreikomponentenreaktion, AgOAc-vermittelte 1,3-dipolare Azomethin-Ylid-Cycloaddition, Yb(PFO)3-vermittelte Dreikomponenten-Pyrazolsynthese) auf ein neues, effizienteres DNA-Kodierungsformat, das den Einsatz von kodierten Startmaterialien ermöglicht. Damit auch harschere Reaktionsbedingungen in der DEL-Synthese verwendet werden können, wird im letzten Teil der Arbeit durch den Austausch der vulnerablen Purinnukleobase Adenin durch die chemisch modifizierte Base 7-Deazaadenin die Etablierung eines chemisch stabilisierten DNA-Barcodes beschrieben.2021-01-01T00:00:00ZStrukturbasierte Entwicklung und Evaluierung von Sondenmolekülen zur allosterischen Regulation von Isoformen der Proteinkinase Akt
http://hdl.handle.net/2003/40510
Title: Strukturbasierte Entwicklung und Evaluierung von Sondenmolekülen zur allosterischen Regulation von Isoformen der Proteinkinase Akt
Authors: Quambusch, Lena
Abstract: Als zentraler negativer Regulator der Apoptose ist die Proteinkinase Akt mit ihren drei Isoformen (Akt1/Akt2/Akt3) entscheidend für das Überleben der Zelle und eine Schlüssel-Zielstruktur in der Wirkstoffforschung. Für ein anhaltendes Scheitern klinischer Akt Wirkstoff-Kandidaten im Kontext der gezielten Krebstherapie mag der Mangel an Informationen über die Akt Isoformen und ihrer pathophysiologischen Rolle in humanen Krankheitsbildern mitverantwortlich sein. Das komplexe Netzwerk der homologen Proteinkinasen konnte bislang anhand von molekular-biologischen Methoden nicht eindeutig aufgelöst werden. Einschnitte in das Interaktom durch genetische Entfernung von Akt ist überschattet durch Redundanzen der anderen homologen Isoform oder mögliche Kompensierungsprozesse über alternative Signalwege. Es bedarf einer gezielten temporalen Kontrolle der Funktionen ebendieser Enzyme, um verknüpft Interaktionsprofile in einer physiologisch-relevanten Zeitskala zu evaluieren. Diese Pertubationsstudien können elementare Grundsteine für neue innovative therapeutische Ansätze liefern sowie dabei helfen, toxische Nebenwirkungen der bisher bekannten Wirkstoffe einzuordnen und diese Vorteile in vielversprechende Behandlungsstrategien zu übersetzen.
Die allosterische Adressierung einer Interdomänen-Bindetasche in Akt mit pharmakologisch-vorteilhaften kovalenten Liganden erwies sich als äußerst vielversprechend. Besonders in Bezug auf Affinität und Selektivität gegenüber ATP-kompetitiven Inhibitoren. Bereits identifizierte allosterische Verbindungen wiesen ein interessantes Selektivitätsprofil gegenüber den Akt Isoformen auf, welches in Kombination mit einer irreversiblen Alkylierungs-Strategie deutlich verbessert werden konnte. Ausgehend von diesen Einblicken in die Struktur-Aktivitätsbeziehung der jeweiligen vermeintlich Isoform-selektiven Liganden sollten weitere Optimierungen durchgeführt werden, um sehr potente, funktionelle Sondenmoleküle zu gewinnen.
Die eingeschränkte Verfügbarkeit von Akt2 und Akt3 Kristallstrukturen konnte mithilfe von Homologiemodellen und detailliertem Sequenzvergleich überwunden werden, um grundlegende Informationen für ein gezieltes Design allosterischer Liganden zu erlangen. Aus den modell-abgeleiteten Bedingungen wurde eine Substanzbibliothek strukturbasiert entworfen und unter Verwendung effizienter synthetischer Strategien umgesetzt. Auf Basis von 50 kovalent-allosterischen Akt Inhibitoren (CAAI) konnten anhand biochemischer Daten Rückschlüsse auf die Struktur-Aktivitätsbeziehung der Liganden gezogen werden. Infolgedessen gelang eine detaillierte Aufschlüsselung von vermeintlichen Bindepräferenzen der allosterischen Akt Isoform-Bindetaschen samt Offenlegung umfangreicher Selektivitätsprofilen der Inhibitoren. Ferner konnten die irreversiblen Eigenschaften der kovalenten Verbindungen ergründet sowie neue strukturelle Einblicke in die Protein-Ligand-Wechselwirkung mithilfe von Röntgenstrukturanalyse gewonnen werden.
Weiterführend konnten die biochemisch erfassten Selektivitätsprofile der Inhibitoren in ein Akt-Isoform abhängiges Zellsystem übersetzt werden, welches es erlaubt, im Hochdurchsatz und ohne Einschränkung durch gewebsspezifische Expressionslevel die Aktivität der Liganden zu bewerten. Außerdem ermöglichte die Identifizierung von Inhibitoren mit optimalen Selektivitätsfenstern im Ba/F3-Modell-Systemen tiefergehende Studien mit der komplexeren Krebszelllinie PANC1. Daraus resultierte die Ergründung von vorteilhaften Inhibitor-Konzentrationen, welche eine selektive Adressierung der jeweiligen Akt Isoform in zellulären Systemen gewähren.
Darüber hinaus konnten in dieser Arbeit funktionalisierte Alkin-Sonden aus den Akt2-selektive Pyrazinon CAAIs gewonnen werden. Als proof-of-concept glückte in ersten in gel Fluoreszenz-Studien die gezielte Modifizierung der Alkin-Funktionalität über eine Kupfer-vermittelte Click-Reaktion mit einem Fluorophor. Diesen chemischen Werkzeugen vermag es gelingen, die Funktionen der Akt Isoformen in komplexen Systemen zu entschlüsseln und ihre Aufgabe in pathophysiologischen Zusammenhängen aufzuklären. Neben der elementaren Rolle in funktionellen Studien stellen die erarbeiteten selektiven Liganden potentielle Wirkstoff-Vorläufer dar, die ein hohes Potenzial besitzen Teil einer innovativen Lösung zur erfolgreichen Adressierung der Proteinkinase Akt und ihrer Isoformen im klinischen Kontext zu sein.2021-01-01T00:00:00ZBiochemische und strukturbiologische Evaluierung von MKK7-Ligand Wechselwirkungen
http://hdl.handle.net/2003/40502
Title: Biochemische und strukturbiologische Evaluierung von MKK7-Ligand Wechselwirkungen
Authors: Wolle, Patrik
Abstract: Mitogen-aktivierte Proteinkinase-Signalwege sind in eukaryotischen Zellen von besonderer Bedeutung, da sie einen zentralen Bestandteil der Weiterleitung von extrazellulären Signalen zum Zellkern darstellen. Beim Menschen sind insgesamt vier MAPK-Signalwege bekannt.
In dieser Arbeit wurde im Speziellen der JNK-Signalweg genauer untersucht, der vor allem in Folge von physischen Stimuli wie Hitze, Strahlung und osmotischen Schock sowie Cytokine aktiviert wird. Ebenso ist der JNK-Signalweg in die Entwicklung von Organen während der Embryogenese und in der Entwicklung des Gehirns und des Nervensystems beteiligt. Dies macht den Signalweg zu einem relevanten Ziel für die Wirkstoffforschung. Es konnte bereits gezeigt werden, dass eine direkte Einwirkung auf JNK, zum Beispiel durch selektive JNK-Inhibitoren, viele Nebenwirkungen auslösen kann. Aus diesem Grund sollte in dieser Arbeit die Regulierung eine der beiden Aktivatoren von JNK, die Mitogen aktivierte Protein Kinase Kinase 7 (MKK7), genauer analysiert werden. Hierfür sollte nach Liganden gesucht werden, welche die Aktivität der Kinase innerhalb eines Organismus spezifisch modulieren können, mit dem Ziel, mit Hilfe dieser Substanz Veränderungen in der Funktionsweise des Signalweges, zum Beispiel in den Phosphorylierungsmustern der JNK Substrate, oder in der Morphologie der Zelle, zu detektieren.
Dazu wurde zunächst eine Auswahl von verschiedenen biochemischen Assays hinsichtlich ihrer Eignung auf die Charakterisierung von MKK7-Liganden untersucht und mit einem, auf diese Weise identifizierten, geeignetem System eine Bibliothek niedermolekularer Substanzen durchmustert. Die gefundenen Hits wurden sowohl biochemisch als auch mit Hilfe von massenspektrometrischen Messungen validiert. Der potenteste Inhibitor (Verbindung 22) wurde anschließend in Mäusen auf seine pharmakokinetischen Eigenschaften hin untersucht, und in einem Ansatz mit DRG-Zellen auf seine spezifische Wirksamkeit hin untersucht und zeigte auch hier eine hohe Aktivität wie auch Selektivität.
Zusätzlich wurde Verbindung 22 wie auch eine Reihe weiterer Inhibitoren im Komplex mit MKK7 kokristallisiert, wodurch der Bindungsmodus dieser Inhibitoren aufgeklärt werden konnte.2021-01-01T00:00:00ZDesign, synthesis and biological evaluation of a pseudo natural product compound collection
http://hdl.handle.net/2003/40495
Title: Design, synthesis and biological evaluation of a pseudo natural product compound collection
Authors: Yildirim, Okan
Abstract: Natural product (NP) scaffolds are valuable starting points for the design and synthesis of novel bioactive compounds as employed in biology-oriented synthesis (BIOS). However, the exploration of chemical and biological space by this method is limited by the guiding NPs. Combining NP fragments in an unprecedented not accessible by current biosynthetic pathways could enable the discovery of novel chemical and biological space. Compound classes obtained by the de novo combination of unrelated NP-derived fragments are called pseudo natural products (pseudo NPs).
Based on the concept of synthesis of pseudo NP compound libraries, an enantioselective double 1,3-dipolar cycloaddition was applied to cyclopentadienones to combine two pyrrolidine fragments in an unprecedented manner (Chapter 3). Highly complex tricyclic molecules with up to eight stereocenters were obtained, including a quaternary center. Detailed mechanistic studies revealed that the double cycloaddition proceeds via an enantioselective dynamic covalent process. In dynamic covalent chemistry (DCC) the reaction follows a thermodynamically controlled pathway in an equilibrium process, allowing the interconversion of products. Time-dependent analysis showed kinetically controlled and thermodynamically favored product formation and an enantioselective interconversion of stereoisomers. Conditions for the selective synthesis of thermodynamically and kinetically controlled structurally complex double cycloadducts with high stereoselectivity from a common set of reagents were developed.
Furthermore, a pseudo NP compound library was designed by combining the pyrrolidine scaffold with pyrroloindoline, generating a highly complex polycyclic compound class (Chapter 4). A synthetic strategy was designed employing an asymmetric 1,3-dipolar cycloaddition, indole formation and dearomatization. Excellent diastereoselectivity was achieved through a substrate controlled cyclization process.; Naturstoff-Grundgerüste sind wertvolle Ausgangspunkte für das Strukturdesign und die Synthese von neuen bioaktiven Verbindungen, wie bereits in der Biologie orientierten Synthese (BIOS) durchgeführt. Die Untersuchung des chemischen und des biologischen Raums ist jedoch von der Wahl des Naturstoffes eingeschränkt. Die Kombination von Naturstofffragmenten in neuartiger Weise, welche über biosynthetische Wege unzugänglich ist, könnte die Entdeckung von neuen chemischen und biologischen Räumen ermöglichen. Verbindungsklassen, welche durch Kombination von nicht miteinander verwandten Naturstofffragment erhalten werden, werden als Pseudo-Naturstoffe bezeichnet.
Für die der Synthese von Bibliotheken von Pseudo-Naturstoffen wurde eine enantioselektive doppelte Cycloaddition an Cyclopentadienon durchgeführt, welche eine neuartige Kombination von Pyrrolidin-Fragmenten ermöglichte (Kapitel 3). Hochkomplexe trizyklische Moleküle mit bis zu acht Stereozentren wurden erhalten, wobei eines der Stereozentren ein quaternäres Zentrum ist. Intensive mechanistische Untersuchungen zeigten, dass die doppelte Cycloaddition durch einen dynamischen kovalenten Prozess abläuft. In der dynamischen kovalenten Chemie folgt die Reaktion einem thermodynamisch kontrollierten Reaktionsverlauf in einem Gleichgewichtsprozess, in welcher die Produkte umgewandelt werden. Eine zeitabhängige Analyse zeigte die Bildung von kinetisch und thermodynamisch kontrollierten Reaktionsprodukten und eine enantioselektive Umwandlung der jeweiligen Stereoisomere. Des Weiteren wurden Reaktionsbedingungen für die selektive Synthese von sowohl kinetisch als auch thermodynamisch kontrollierten hochkomplexen Cycloadditions-Produkten mit hoher Stereoselektivität unter Anwendung von gleichen Reagenzien entwickelt.
Des Weiteren wurde eine weitere Pseudo-Naturstoff-Substanzbibliothek durch Kombination von Pyrrolidin-Grundgerüsten mit Pyrroloindolin entwickelt, bei der polyzyklische Verbindungen erhalten wurden. Eine Synthesestrategie aus 1,3-dipolarer Cycloaddition, Indol-Bildung und anschließender Dearomatisierung wurde angewendet. Exzellente Diastereoselektivität wurde durch eine substrat-kontrollierte Zyklisierung erzielt.2021-01-01T00:00:00ZNovel concepts and methodologies in pseudo natural product chemistry
http://hdl.handle.net/2003/40490
Title: Novel concepts and methodologies in pseudo natural product chemistry
Authors: Liu, Jie
Abstract: Natural products (NPs) are an inspiring source for chemical biology and drug discovery research, featuring complex and diverse structures. However, the chemical space explored by nature is limited by the biosynthetic precursors and machineries available in NP-producing organisms. To explore the vast chemical space of biological relevance, pseudo natural product design is proposed to unprecedentedly recombine NP fragments from different sources. This thesis aims to develop novel concepts and methodologies in pseudo natural product design to supply structurally complex and diverse compound collections for biological study. To answer the question whether varying connections between two fragments can lead to diverse biological performance, a systematic recombination of alkaloid-derived NP fragments pyrrolidine and tetrahydroquinoline is disclosed in Chapter 3. Efficient and concise methodologies are developed to afford a library of 123 members and 7 scaffolds varying the connectivity patterns, positions and saturation states. To improve the synthetic efficiency, a previously neglected Rh(III)-catalyzed reaction between benzhydroxamate and diazooxindole is presented in Chapter 4. Besides varying connections between two fragments, ring distortion and pseudo natural product design are conceptually combined where sesquiterpene lactones are converted to diverse scaffolds via ring distortion followed by incorporation of alkaloid-derived pyrrolidine fragments yielding pseudo sesquiterpenoid alkaloids in Chapter 5. To increase the diversity of the compound collection, a stereodivergent 1,3-dipolar cycloaddition is developed to afford stereocomplementary pyrrolidines.2021-01-01T00:00:00ZFGF4 drives intermittent oscillations of ERK activity in mouse embryonic stem cells
http://hdl.handle.net/2003/40484
Title: FGF4 drives intermittent oscillations of ERK activity in mouse embryonic stem cells
Authors: Raina, Dhruv
Abstract: Signal transduction systems often display complex activation patterns in response to steady-state ligand stimulation. It has been hypothesized that cells use signal transduction dynamics to encode information about the concentration or identity of the stimuli, but rigorous testing of this idea requires systematic characterisation of single cell responses in defined environments. Here, I used live-cell sensors to study ERK activity dynamics in mouse embryonic stem cells (ESCs) in response to fibroblast growth factor (FGF) stimulation, the main trigger for ESC differentiation and lineage commitment. I detected previously undescribed rapid and regular pulses of ERK activity in pluripotent ESCs. Using Fgf4 mutant cells, I showed that paracrine FGF was the main driver of ERK pulses. ERK activity dynamics were heterogeneous between cells, ranging from oscillatory to stochastic behaviours. Additionally, single cells showed the ability to transit between oscillatory and non-oscillatory behaviour, leading to intermittent clusters of regular pulsing. With increasing FGF4 dose, clusters of pulses became more prevalent and pulse frequency increased correspondingly, although the duration of individual pulses had a characteristic timescale that was maintained. Increasing FGF4 dose also increased the basal phospho-ERK levels in single cells. The data in this thesis proposes that ligand levels are reflected in the combination of both the basal ppERK level, as well as the pulse frequency.; Zelluläre Signaltransduktionssysteme erzeugen nach Stimulation mit konstanten Konzentrationen von Signalmolekülen oft komplexe dynamische Aktivierungsmuster. Zellen können diese Aktivierungsmuster nutzen, um Informationen über die Konzentration oder Identität der Stimuli zu übertragen. Um die Funktion dynamischer Aktivierungsmuster in spezifischen Zellsystemen zu verstehen, ist es notwendig, Einzelzellantworten in definierten Umgebungen systematisch zu charakterisieren. Der Fibroblasten-Wachstumsfaktor (Fibroblast Growth Factor, FGF) ist ein zentrales Signalmolekül das die Differenzierung pluripotenter embryonaler Stammzellen (embryonic stem cells, ESCs) steuert. In dieser Arbeit habe ich Live-Zell-Sensoren verwendet, um die ERK-Aktivitätsdynamik ESCs der Maus nach FGF-Stimulation zu untersuchen.
Durch meine Experimente konnte ich bisher unbeschriebene schnelle und regelmäßige Pulse der ERK-Aktivität in ESCs nachweisen. Mithilfe von Fgf4-mutierten Zellen konnte ich zeigen, dass diese ERK-Pulse im Wesentlichen durch parakrine FGFs ausgelöst werden. Die Dynamik der ERK-Aktivität in ESC-Kulturen ist heterogen und reicht von oszillatorischem bis zu stochastischem Verhalten. Einzelne Zellen zeigen außerdem die Fähigkeit, zwischen oszillatorischem und nicht-oszillatorischem Verhalten zu wechseln, ein Verhalten das sich im Auftreten von Clustern von regelmäßigen Pulsen niederschlägt. Solche Cluster treten mit zunehmender FGF4- Dosis vermehrt auf, und die Pulsfrequenz nimmt entsprechend zu, wobei die Dauer der einzelnen Pulse allerdings eine charakteristische Zeitskala hat die unabhängig von der FGF-Dosis ist. Mit zunehmender FGF4-Dosis steigt zudem der basalen phospho- ERK-Spiegel in einzelnen Zellen an. Zusammen legen diese Daten nahe, dass das FGF/ERK Signaltransduktionssystem in ESCs Information über die Konzentration von FGF-Liganden in einer Kombination aus basalem ppERK-Spiegel und ERK- Pulsfrequenz überträgt.2021-01-01T00:00:00ZDesigner receptors for nucleotide-resolution analysis of genomic 5-Methylcytosine by cellular imaging
http://hdl.handle.net/2003/40364
Title: Designer receptors for nucleotide-resolution analysis of genomic 5-Methylcytosine by cellular imaging
Authors: Muñoz-López, Álvaro; Buchmuller, Benjamin; Wolffgramm, Jan; Jung, Anne; Hussong, Michelle; Kanne, Julian; Schweiger, Michal Ruth; Summerer, Daniel
Abstract: We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution.2020-03-13T00:00:00ZTEAD-YAP interaction inhibitors and MDM2 binders from DNA-encoded indole-focused Ugi-peptidomimetics
http://hdl.handle.net/2003/40334
Title: TEAD-YAP interaction inhibitors and MDM2 binders from DNA-encoded indole-focused Ugi-peptidomimetics
Authors: Kunig, Verena B. K.; Potowski, Marco; Akbarzadeh, Mohammad; Škopić, Mateja Klika; Santos Smith, Denise dos; Arendt, Lukas; Dormuth, Ina; Adihou, Hélène; Andlovic, Blaž; Karatas, Hacer; Shaabani, Shabnam; Zarganes-Tzitzikas, Tryfon; Neochoritis, Constantinos G.; Zhang, Ran; Groves, Matthew; Guéret, Stéphanie M.; Ottmann, Christian; Rahnenführer, Jörg; Fried, Roland; Dömling, Alexander; Brunschweiger, Andreas
Abstract: DNA-encoded combinatorial synthesis provides efficient and dense coverage of chemical space around privileged molecular structures. The indole side chain of tryptophan plays a prominent role in key, or “hot spot”, regions of protein–protein interactions. A DNA-encoded combinatorial peptoid library was designed based on the Ugi four-component reaction by employing tryptophan-mimetic indole side chains to probe the surface of target proteins. Several peptoids were synthesized on a chemically stable hexathymidine adapter oligonucleotide “hexT”, encoded by DNA sequences, and substituted by azide-alkyne cycloaddition to yield a library of 8112 molecules. Selection experiments for the tumor-relevant proteins MDM2 and TEAD4 yielded MDM2 binders and a novel class of TEAD-YAP interaction inhibitors that perturbed the expression of a gene under the control of these Hippo pathway effectors.2020-06-14T00:00:00ZA biochemical pacemaker system for robust actin growth
http://hdl.handle.net/2003/40169.2
Title: A biochemical pacemaker system for robust actin growth
Authors: Funk, Johanna
Abstract: Eukaryotic cells determine their shape and organize their interior through a dynamic actin cytoskeleton that forms a variety of architectures, each crucial for a unique cellular function. Common to all actin structures is their growth from soluble subunits that assemble into filaments. The assembly of functional actin networks requires control over the speed at which filaments grow. How this can be realized at the high but variable concentrations of soluble actin subunits found in cells is not known. Biochemical reconstitution has singled out the concentration of soluble actin subunits as the key factor that directly controls the speed of actin growth in a linear manner. At the same time, however, this creates a paradox: The soluble actin concentration differs strongly not only between organisms, but also among distinct cell types and likely even in distinct locations within a single cell. Our current view of actin assembly posits that these large differences will result in dramatically different actin growth rates. We presently do not understand how cells deal with changes in the concentration of polymerizable actin to control filament growth. Here we develop new methods to visualize actin growth at single filament resolution over concentrations previously simply inaccessible. This puts us in the unique position to reconstitute actin assembly over the full physiological range of free subunit levels for the first time. Using this novel approach, we discover that under cell-like conditions, actin growth is not controlled by the concentration of soluble subunits. Instead, we identify a key reaction in the actin elongation cycle the release of the monomer-binding protein profilin from the filament end- as the kinetic bottleneck that limits the speed of filament growth. We show that this kinetic limit we discovered in vitro confers robustness to actin growth in the cytoplasm of mammalian cells. The fundamental finding is that this mechanism buffers the speed of actin filament growth against changes in the soluble subunit concentration, an essential requirement for the control of actin dynamics in cells. This opens a myriad of new opportunities to understand the regulation of actin dynamics and we explore the most immediate consequences. We show that the kinetic limit to actin growth imposed by profilin is not fixed but actively modulated by actin polymerases: Formin proteins, previously thought to simply increase the concentration of actin subunits at the actin filament end, catalytically promote the release of profilin from actin. This transforms our mechanistic understanding of these essential actin polymerases. In general terms, we show how the collective action of formin and profilin constitutes a molecular pacemaker system that creates robust, but tunable rates of actin growth.2021-01-01T00:00:00ZPrediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations
http://hdl.handle.net/2003/40290
Title: Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations
Authors: Albrecht, Wiebke
Abstract: Drug-induced liver injury (DILI) is the leading cause for acute liver failure in the USA and in Germany and one of the most common reasons for withdrawal of drugs from the market or failure of a drug candidate during development. Since DILI cannot be accurately predicted by animal models, a reliable in vitro test system for the prediction of human hepatotoxicity would be a valuable asset for drug development as well as for regulatory purposes.
In this thesis an in vitro/in silico approach for the prediction of human hepatotoxicity in relation to blood concentrations and oral doses was established. This approach combines in vitro effective concentrations derived from a cytotoxicity assay, in vivo concentrations obtained by physiologically based pharmacokinetic (PBPK) modelling and a support vector machine (SVM) classifier based on these concentration pairs to predict the risk for hepatotoxicity for specific exposure scenarios. For systematic test system evaluation and optimization two novel performance metrics, the Toxicity Separation Index (TSI) and Toxicity Estimation Index (TEI), were utilized. These indices eliminate the need for a priori defined in vitro and in vivo concentrations and foster the systematic evaluation of the benefit of additional readouts.
As a first step the feasibility of the in vitro/in silico approach was tested for primary human hepatocytes (PHH) and a training set of 28 compounds with in total 30 different in vitro/in vivo concentration vectors, yielding a sensitivity of 100%, a specificity of 88% and an accuracy of 93% in the leave-one-out classification with the SVM based classifier. A SVM based classifier utilizing all vectors was then applied to derive in combination with reverse PBPK modelling an acceptable daily intake (ADI) for the hepatotoxicant pulegone. The derived ADI was comparable to published ADIs based on two rodent studies.
Next, the compound set was extended to a total of 80 compounds with 82 distinct in vitro/in vivo concentration pairs. The SVM leave-one-out classification resulted in a sensitivity of 77.8%, a specificity of 59.4% and an accuracy of 70.1%.
Furthermore, the feasibility of the approach substituting HepG2 cells for the PHH and a combination of both cell culture systems for the extended compound set was evaluated. The obtained sensitivity was 88.9% and 86.7% and the specificity 62.5% and 65.6%, respectively. The accuracy was in both cases 77.9%.2021-01-01T00:00:00ZEngineered TALE repeats for enhanced imaging-based analysisof cellular 5-Methylcytosine
http://hdl.handle.net/2003/40205
Title: Engineered TALE repeats for enhanced imaging-based analysisof cellular 5-Methylcytosine
Authors: Muñoz‐López, Alvaro; Jung, Anne; Buchmuller, Benjamin; Wolffgramm, Jan; Maurer, Sara; Witte, Anna; Summerer, Daniel
Abstract: Two‐TALE test: Engineered transcription‐activator‐like effector (TALE) repeats can be used to design programmable receptors for improved in situ imaging‐based analysis of 5 mC in user‐defined DNA sequences. Pairs of natural 5 mC‐discriminating and engineered 5 mC‐binding TALEs allow nucleotide resolution analysis of 5 mC with response of both TALEs. This provides an impulse for the design of programmable imaging probes for studying 5 mC functions in chromatin regulation with increased dynamic range.2020-09-20T00:00:00ZStruktur-basiertes Design, Synthese und pharmakokinetische Charakterisierung von kovalenten Inhibitoren zur Adressierung von Krebs-relevanten Proteinkinasen
http://hdl.handle.net/2003/40201
Title: Struktur-basiertes Design, Synthese und pharmakokinetische Charakterisierung von kovalenten Inhibitoren zur Adressierung von Krebs-relevanten Proteinkinasen
Authors: Hardick, Julia
Abstract: Die vorliegende Arbeit beinhaltet zwei Abschnitte: (i) die pharmakokinetische Charakterisierung von Inhibitoren mit dem Fokus auf der Analyse der metabolischen Stabilität in Anwesenheit von Lebermikrosomen, die einen Phase I Metabolismus nachbilden. Hierzu wurde ein entsprechendes Assaysystem in der Arbeitsgruppe etabliert. (ii) Das Struktur basierte Wirkstoffdesign, die organische Synthese von niedermolekularen Verbindungen und deren Charakterisierung, mit dem Ziel selektive und potente Inhibitoren für Krebs relevante Proteinkinasen zu identifizieren. Die Kombination aus biochemischem Aktivitätsassay und zellulärem Viabilitätsassay erlaubte eine Bewertung der generierten Substanzbibliotheken. Massenspektrometrische Studien ermöglichten die Analyse der kovalenten Adressierung des Zielproteins, sowie Komplexkristallstrukturen verwendet wurden, welche tiefere Einblicke in die Bindungsmodi der Inhibitoren erlaubten.
Im Rahmen dieser Arbeit wurde ein Phase I Metabolismus Assays etabliert, der einen wichtigen Beitrag zur Optimierung und Weiterentwicklung der Inhibitoren leistet. Der Assay wurde in Gegenwart von humanen und murinen Lebermikrosomen durchgeführt, welche die Cytochrom P450 Enzyme beinhalten, durch die die Mehrheit der auf dem Markt zugelassenen Wirkstoffe abgebaut werden. Untersucht wurde die metabolische Stabilität von 200 Verbindungen über die Zeit, sodass Parameter wie die Halbwertszeit (t1/2) und die intrinsische Clearance (CLint) für eine Vielzahl an Verbindungen aus unterschiedlichen Projekten bestimmt und mit Referenzverbindungen verglichen werden konnten.
Die Proteinkinase MKK7 ist Teil des JNK Signalwegs, dessen Fehlregulierung in neurodegenerativen Erkrankungen sowie bei Herzinfarkten mit darauffolgenden ischämischen Reperfusionsschäden identifiziert wurde. Unzureichende Selektivitätsprofile und mangelnde Wirksamkeit wurden bislang für JNK Inhibitoren beschrieben, weshalb die Adressierung der JNK aktivierenden Kinase MKK7 eine gute Alternative darstellt die genannten Selektivitätsmängel zu umgehen. Der literaturbeschriebene, potente Pyrazolopyrimidin-basierte kovalente MKK7 Inhibitor 24 sollte in komplexen zelluläre in vitro und ersten in vivo Modellsystemen getestet werden, weshalb zunächst ein Gramm der Verbindung hergestellt wurde. Die Identifizierung einer geeigneten Formulierung für die Substanz sowie die Bestimmung der maximal tolerierbaren Dosis bildeten die Grundlage für erste in vivo Mausstudien. Aus diesen ging bei 600 mg pro kg Körpergewicht der Maus durch IV Applikation eine maximale Plasmakonzentration von 5 µM hervor, sowie eine gute Verträglichkeit der Substanz. Die nur mäßige Löslichkeit der Verbindung stellte sich als problematisch dar, weshalb verschiedene Salzformen dargestellt wurden, von denen das HCl Salz 34 eine verbesserte Löslichkeit zeigte. Weiterhin wurde der Einfluss von 24 auf Reperfusionsschäden infolge eines Herzinfarktes in Mausmodellen untersucht.
Hierbei wurden Herzinfarkte induziert und durch die präventive Vorab Gabe der entstandene ischämische Reperfusionsschaden im Vergleich zu unbehandelten Mäusen analysiert. Basierend auf den bislang erzielten Ergebnissen lässt sich ein geringerer ischämischer Reperfusionsschaden bei vorheriger Verabreichung von 24 vermuten.
Die Behandlung von Krebs wurde in den letzten Jahren durch den Ansatz der Präzisionsmedizin revolutioniert. Hierbei können durch die Identifizierung von prädiktiven Biomarkern große Patientenpopulationen in Subgruppen unterteilt werden, die somit eine individuelle und maßgeschneiderte Therapie erhalten. Die zielgerichtete Adressierung mit TKIs wurde erfolgreich für die Proteinkinasen EGFR, KIT und PDGFR beschrieben.
Her2, ein Mitglied der ErbB Rezeptorfamilie, wurde bei etwa 4 % aller Patienten mit NSCLC als Treiber identifiziert, wovon ca. 80 % Insertionsmutationen in Exon20 entsprechen. Geringe Überlebens und Ansprechraten von < 40 % auf die bislang angewendeten Therapien mit TKIs verdeutlichen den dringenden Bedarf an wirksamen Inhibitoren für Krebspatienten mit positivem Her2 Mutationsstatus. In dieser Arbeit wurden kovalente, Pyrrolopyrimidin basierte Typ II Inhibitoren mit dem Ziel weiterentwickelt, die Löslichkeit der Inhibitoren durch Einführung von Löslichkeitsgruppen zu steigern und somit eine verbesserte zelluläre Aktivität erzielen zu können. Durch Einbringung von polaren Alkohol und Amin-funktionalitäten in ortho Position zum Michael Akzeptor wurde eine gesteigerte biochemische, aber gleichbleibende zelluläre Aktivität festgestellt. Kristallisationsstudien verifizierten den angenommenen Bindungsmodus und die pharmakokinetische Charakterisierung zeigte bspw. Optimierungsbedarf bzgl. der Stabilität in murinem Blutplasma sowie in Lebermikrosomen.
Mutationen in den Proteinkinasen KIT und PDGFR wurden bei der Mehrheit von Patienten mit gastrointestinalen Stromatumoren identifiziert. Die derzeitig zugelassenen Wirkstoffe für die Behandlung von GIST sind reversible, ATP kompetitive Inhibitoren, von denen lediglich Avapritinib und Ripretinib speziell für die Therapie von GIST entwickelt wurden. Im Rahmen dieser Arbeit wurde basierend auf der Ponatinibstruktur eine Substanzbibliothek von Typ II bzw. Typ III Inhibitoren synthetisiert. Diese wurden mit einer reaktiven Gruppe dekoriert, um eine kovalente Bindung mit Cys788 oder Cys809 in KIT bzw. Cys814 oder Cys835 in PDGFR auszubilden. In MS und MS/MS Experimenten konnte die spezifische Bindung der synthetisierten Inhibitoren an Cys788 in KIT nicht nachgewiesen werden. Ferner wurden einzigartige, Patienten abgeleitete PDGFR mutierte Zelllinien entwickelt und in einem HTS eingesetzt. Die Durchmusterung von fokussierten Kinaseinhibitor-bibliotheken sollte zukünftig neue Leitstrukturen identifizieren, um spezifische Inhibitoren für PDGFR mutierte gastrointestinale Stromatumore designen und synthetisieren zu können.2021-01-01T00:00:00ZPseudo-natural products via Pictet-Spengler reaction and dearomatization
http://hdl.handle.net/2003/40200
Title: Pseudo-natural products via Pictet-Spengler reaction and dearomatization
Authors: Burhop, Annina
Abstract: Natural products (NPs) occupy a broad diversity in bioactivity and therefore, serve as a comprehensive inspiration in drug discovery. Many design approaches for small molecule collections are based on NPs while aiming for extending nature’s chemical space to discover unexpected or novel bioactivities. Analogously, the pseudo-NP approach affords novel scaffolds by combining unrelated NP fragments in complexity-generating reactions.
A collection of spirocyclic pseudo-NPs was generated via iso-oxa-Pictet-Spengler reaction, for which new conditions were developed. Cheminformatic analysis indicated the pseudo-NPs to cover chemical space sparsely represented by NPs, drugs and drug-like compounds and that they may combine favorable properties of these compound classes. Biological evaluation of indofulvins derived from 2-hydroxyethyl-indoles and the fragment-sized NP griseofulvin in different cell-based assays and the high content cell painting assay revealed them to define a new autophagy inhibitor chemotype that targets mitochondrial respiration.
A new level of complexity and diversity was introduced by the dearomatization of indole-containing pseudo-NPs. The ɣ-pyrone annulation was optimized to yield structural diverse indolenines and indolines that show biological differences to their parent compounds in the cell painting assay.
Morphological profiling was furthermore employed to identify relevant chemical structures for bioactivity to potentially guide future synthesis of small compound collections. These results suggest that the pseudo-NP concept in combination with the cell painting assay may be able to be used to rationally design new compound classes and therefore, further explore chemical space with biological relevance.2020-01-01T00:00:00ZMorphological profiling of small molecules for mode-of-action studies using the Cell Painting Assay
http://hdl.handle.net/2003/40178
Title: Morphological profiling of small molecules for mode-of-action studies using the Cell Painting Assay
Authors: Schneidewind, Tabea
Abstract: The Cell Painting Assay (CPA) is an unbiased morphological profiling approach that generates a holistic view of the bioactivity space. Therefore, it is a powerful tool to deconvolute targets or mode-of-action (MoA) of small molecules, which is especially important for non-protein targets as they are difficult to identify with commonly applied methods.
In order to examine the applicability of the CPA to identify non-proteins targets, the morphological fingerprint of the iron chelator Deferoxamine (DFO), as well as references and so far uncharacterized compounds with similar fingerprints, were investigated. Reference compounds, biosimilar to DFO, possess different annotated targets and activities but share a common MoA of cell cycle arrest. This was experimentally confirmed for a representative selection of references. The cluster analysis enabled the identification of novel and so far uncharacterized chelating agents and DNA synthesis modulators. Furthermore, hierarchical clustering, using the CPA fingerprints, revealed a first insight into the different mechanisms of action.
To investigate the bioactivity of a small tetrahydroindolo[2,3-a]quinolizine compound class, a combination of morphological profiling using the CPA and proteome profiling was pursued. The results revealed an altered cholesterol homeostasis induced by the compound’s physicochemical properties that led to an accumulation and an increased pH in lysosomes. More than 400 reference compounds and well-characterized drugs with different annotated targets and activities share high biosimilarity to the most active derivative. The majority of the compounds in this cluster also possess physicochemical properties, that are predictive for the accumulation in lysosomes. Modulation of cholesterol homeostasis was experimentally confirmed for a representative selection of references. Therefore, this cluster can be used to identify novel modulators of cholesterol homeostasis but also to associate the regulation of corresponding genes or proteins to an effect induced by the physicochemical properties of the compounds.
The findings presented in this thesis emphasize the power of the CPA to evaluate bioactive small molecules and to predict diverse MoA for uncharacterized compounds as well as to uncover and expand so far unknown activity for already characterized small molecules and drugs.2021-01-01T00:00:00ZRole of histone modifications in the recruitment of remodeling complex RSC and lysine deacetylase Hst2 to chromatin
http://hdl.handle.net/2003/40130
Title: Role of histone modifications in the recruitment of remodeling complex RSC and lysine deacetylase Hst2 to chromatin
Authors: Jain, Neha
Abstract: Long DNA molecule must not just be somehow stored within a tiny cell but also be accessible by protein machineries to allow for timely execution of key cellular events such as chromatin compaction, cell division, transcription, etc. My PhD aimed at identifying the Histone protein modifications that recruit proteins to DNA using genetically encoded unnatural amino acid mutagenesis. For example, using pBPA as a crosslinker in budding yeast, I identified a novel preference of the RSC Chromatin Remodeler to H2B SUMOylated nucleosomes. This paves way for a better understanding into the intricate recruitment mechanisms of RSC to its substrate. In another project, I investigated the molecular mechanism behind the histone modification H3 S10 phosphorylation mediated chromosome compaction in mitosis. I showed that the interaction of Lysine Deacetylase, Hst2 with H3 S10ph is dependent on the 14-3-3 protein Bmh1. This phosphorylated Hst2-Bmh1 interaction is attuned with the cell cycle reaching its maximum in M phase. I could recapitulate the binding event in vitro by purifying genetically encoded phosphorylated Hst2. Furthermore, I also demonstrated that phosphorylation at C-terminal residues of Hst2 enhances its deacetylase activity releasing a previously identified autoinhibition of the enzyme. Hence, the data presented here address key mechanisms of chromosome compaction in mitosis.2020-01-01T00:00:00ZStructural investigations on cholesterol binding membrane proteins SREBP cleavage-activating protein (Scap) and Patched1 by cryo-EM
http://hdl.handle.net/2003/40129
Title: Structural investigations on cholesterol binding membrane proteins SREBP cleavage-activating protein (Scap) and Patched1 by cryo-EM
Authors: Weyers, Birte
Abstract: Membrane proteins are essential for cell viability, as they are responsible for numerous regulatory processes like cell-cell communication, signal transduction or the transport of molecules across lipid bilayers. Furthermore, mutations in their amino acid sequence or altered three-dimensional (3D) conformations can lead to severe diseases. Based on their significant role, membrane proteins represent the majority of all drug targets (more than 60%). For a complete understanding of their function, the 3D structure of the protein is necessary and provides a first insight on a molecular level that furthermore enables the research for new efficient drugs. In this thesis, structural investigations of two polytopic membrane proteins, called Patched and sterol regulatory element binding protein- (SREBP-) cleavage activating protein (SCAP), were performed. Both membrane proteins bind cholesterol and contain the sterol sensing domain (SSD) at the transmembrane helix 2 to 6 which is highly conserved upon membrane proteins involved in cholesterol metabolism.
The first membrane protein, Patched, is one of the key proteins in Hedgehog (Hh) signalling, which is one of the main pathways in tissue patterning events during embryonic development and stem cell biology. Patched suppresses Smoothened (Smo) activity by an unknown mechanism that leads to an inactivation of the Hh signalling cascade which is lifted upon the complex formation of Patched and Hh proteins. Here, I show the expression and purification of a C-terminally truncated Patched1 construct with a yield of 0.01 mg protein per liter of mammalian cell culture which was sufficient and suitable for structural analysis by transmission electron microscopy (EM). However, before I had the chance to structurally characterize Patched1, several cryo-EM structures of mammalian Patched1 alone and in complex with Sonic Hh (Shh) were reported by other groups in the last two years.
The membrane protein Scap regulates the cholesterol homeostasis via escorting the transcriptional regulator SREBP from the endoplasmic reticulum (ER) to the Golgi in a sterol dependent manner. At increased cholesterol concentrations, the sterol is bound to Scap and prevents the Scap-SREBP complex movement to the Golgi, as well as the proteolytic processing of SREBP. Although the different complex states of Scap are of great interest, the structure of the full-length Scap is still remaining. In this thesis, I present the first cryo-EM reconstruction of the monomeric full-length mammalian Scap at sub-nanometer resolution. A previously determined crystal structure of the C-terminal WD40 domain from fission yeast Scap could be unambiguously docked into this reconstruction. I therefore predict a similar structure of the mammalian WD40 repeat domain, which forms an eight-bladed β-propeller. These data provide a first insight into the architecture of the full-length mammalian membrane protein Scap and can be very valuable for structure analysis of Scap complex formations.2021-01-01T00:00:00ZSequence-specific DNA binders for nucleotide resolution analysis genomic 5-methylcytosine by cell imaging
http://hdl.handle.net/2003/40128
Title: Sequence-specific DNA binders for nucleotide resolution analysis genomic 5-methylcytosine by cell imaging
Authors: Munoz-Lopez, Alvaro
Abstract: 5-methylcytosine (5mC) is a fundamental epigenetic modification in mammalian genomes involved in development, cell differentiation and genomic imprinting. In addition, aberrant DNA methylation patterns are responsible for the pathogenesis of many diseases including neurodegenerative disorders, cardiovascular affections and cancer. In this thesis, we describe the development of a novel method for image-based analysis of 5mC using pairs of fluorescent Transcription Activator Like-Effectors (TALEs). These DNA binding proteins can recognize specific sequences of canonical or epigenetically modified DNA via modular repeats that interact with nucleobases in a one-to-one correspondence. We employed fluorescent TALE pairs that differ only in the repeat responsible for recognizing cytosine (C) at CpG dinucleotides and the fluorophore fused to them (either eGFP or mCherry). By using the 5mC selective repeat HD in one of the TALEs, we can detect differences in methylation level, while the universal binder repeat G* in the other TALE is not responsive to 5mC and allows to detect local changes in chromatin compaction. This way it is possible to analyze 5mC independently of potential differences in target accessibility. We applied our method using recombinantly expressed and purified TALE pairs in cellular stains to image SatIII DNA. This pericentromeric DNA is the origin of nuclear stress bodies (nSBs), exhibits aberrant methylation in several cancers and remains challenging to study by conventional methods due to its highly repetitive nature. We proved the applicability of our method to study 5mC differences in user-defined repetitive sequences with single nucleotide and strand resolution. Furthermore, we correlated the methylation status of SatIII with the presence of heat shock factor 1 (HSF1) at its recognition sequence after stress, revealing a role for 5mC in HSF1 recruitment as initial step of nSB formation in a subpopulation of cells. Finally, we constructed and screened a library of size-reduced TALE repeats to identify potential 5mC binders. We found that RVD NH* binds selectively to 5mC, but not C and its application in combination with HD TALEs allows for improved imaging with higher dynamic range. These studies offer a promising imaging tool for studying 5mC function in repetitive sequences and its interplay with other imageable chromatin-interacting proteins with nucleotide, strand, locus and cell resolution.2020-01-01T00:00:00ZKristallisation und strukturbiologische Charakterisierung klinisch-relevanter Mutationsvarianten der Rezeptor-Tyrosinkinasen EGFR und Her2
http://hdl.handle.net/2003/40111
Title: Kristallisation und strukturbiologische Charakterisierung klinisch-relevanter Mutationsvarianten der Rezeptor-Tyrosinkinasen EGFR und Her2
Authors: Niggenaber, Janina
Abstract: Lungenkrebs ist die häufigste Ursache für krebsbedingte Todesfälle weltweit.[1] In den letzten Jahren hat jedoch die sogenannte Präzisionsmedizin die Krebstherapie von Nicht-kleinzelligen Lungenkrebs-Patienten (NSCLC-Patienten) revolutioniert. Die Identifizierung von prädikativen Biomarkern sowie das detaillierte genetische Verständnis ermöglichte die Entwicklung niedermolekularer Verbindungen zur gezielten Inhibierung der aberranten Zielstrukturen genetisch definierter Patientengruppen. Durch die gezielte Adressierung der onkogenen Zielproteine können die Nebenwirkungen verringert werden, wodurch die Präzisionsmedizin eine vielversprechende Alternative zur platinbasierten Chemotherapie darstellt.[2,3]
Genetische Mutationen, die zur Entstehung und zur Progression von NSCLC führen, sind häufig in den Rezeptor-Tyrosinkinasen zu finden. Hierbei sind im Rahmen dieser Arbeit vor allem der epidermale Wachstumsfaktor-Rezeptor (EGFR) sowie der humane epidermale Wachstumsfaktor-Rezeptor 2 (Her2) zu nennen.[4,5] Die Entstehung von Resistenzmutationen innerhalb der Kinase-Domäne von EGFR, während der Behandlung mit zielgerichteten Tyrosinkinase-Inhibitoren, erfordert das stetige Aufklären der Resistenzmechanismen sowie die Entwicklung neuer Wirkstoffe.[6] Daher ist ein detailliertes Verständnis der Mutanten sowie der Protein-Inhibitor-Interaktionen auf molekularer Ebene für die Entwicklung neuartiger Verbindungen erforderlich.
Die Proteinkristallographie stellt eine wichtige Methode zur Strukturaufklärung dar. [7,8] Allerdings sind bis zur finalen Kristallstruktur viele Herausforderungen wie das Konstruktdesign, die Proteinexpression in unterschiedlichen Expressionssystemen sowie die Proteinreinigung und die Identifizierung geeigneter Kristallisation-bedingungen zu meistern.
Nach Optimierung der genannten Arbeitsschritte konnten im Rahmen dieser Arbeit verschiedene Kristallisationssysteme Krebs-relevanter EGFR-Varianten etabliert werden, mit deren Hilfe ein verlässliches Wachstum qualitativ hochwertiger Proteinkristalle in Komplex mit niedermolekularen Verbindungen für die Strukturaufklärung ermöglicht werden konnte. Neben dem Interaktionsnetzwerk der Verbindungen und es Zielproteins konnten wichtige strukturelle Einblicke für weitere strukturbasierte Designansätze neuartiger Inhibitoren sowie Optimierungen bereits vorliegende Inhibitoren identifiziert werden.
Im Rahmen dieser Arbeit konnten insgesamt 47 finale Kristallstrukturen der EGFR-Mutationsvarianten generiert werden, von denen bisher zwölf in der Protein Data Bank publiziert wurden.
[1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, A. Jemal. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. Ca: Cancer J. Clin. 2018, 68, 394-424. [2] A. Thomas, S. V. Liu, D. S. Subramaniam, G. Giaccone. Refining the Treatment of NSCLC According to Histological and Molecular Subtypes. Nat. Rev. Clin. Oncol. 2015, 12, 511-526. [3] S. O. Dolly, D. C. Collins, R. Sundar, S. Popat, T. A. Yap. Advances in the Development of Molecularly Targeted Agents in Non-Small-Cell Lung Cancer. Drugs 2017, 77, 813-827. [4] S. V. Sharma, D. W. Bell, J. Settleman, D. A. Haber. Epidermal Growth Factor Receptor Mutations in Lung Cancer. Nat. Rev. Cancer 2007, 7, 169-181. [5] J. Mendelsohn, J. Baselga. The EGF Receptor Family as Targets for Cancer Therapy. Oncogene 2000, 19, 6550-6565. [6] J. Lategahn, M. Keul, D. Rauh. Lessons To Be Learned: The Molecular Basis of Kinase-Targeted Therapies and Drug Resistance in Non-Small Cell Lung Cancer. Angew. Chem., Int. Ed. 2018, 57, 2307-2313. [7] M. W. Parker. Protein Structure from X-Ray Diffraction. J. Biol. Phys. 2003, 29, 341-362. [8] Z. Sayers, B. Avsar, E. Cholak, I. Karmous. Application of Advanced X-Ray Methods in Life Sciences. Biochim. Biophys. Acta. Gen. Subj. 2017, 1861, 3671-3685.2021-01-01T00:00:00ZA role of glycerophosphodiesterase EDI3 in glycogen metabolism
http://hdl.handle.net/2003/40096
Title: A role of glycerophosphodiesterase EDI3 in glycogen metabolism
Authors: Leonhardt, Gregor M.
Abstract: The glycerophosphodiesterase EDI3 (endometrial differential 3) is a key enzyme in various metabolic signalling pathways because it cross-links the triglyceride and choline signalling pathway by hydrolysing glycerophosphocholine (GPC) to glycerol-3-phosphate (G3P) and choline. The enzyme has two functional domains - the GDE domain, where the active site of the protein is located, and the carbohydrate binding domain (CBM), whose function is completely unknown. EDI3 is part of the evolutionary conserved GDE protein family, whose members have different functions. In previous work, high EDI3 expression in primary toumors of ovarian and endometrial carcinomas was associated with increased metastasis, increased cell migration and poorer survival. In this work the role of the CBM20 domain was investigated on a large scale and a possible new role for EDI3 was found.
The present work confirms previous research on other CBM20 proteins (Laforin and STBD1) that showed that the CBM domain is essential for protein stability. The deletion of the entire CBM20 domain (ΔCBM20) and mutations of a conserved amino acid within the CBM20 (W32A) significantly reduced protein stability. As a consequence of these mutations, the enzymatic activity of the protein was decreased (W32A) or completely eliminated (ΔCBM20) and the phenotypic migration effect was reduced. Furthermore, this work could show that EDI3 forms dimers or oligomers and the CBM20 domain is required for this process.
A characteristic of CBM20 is the interaction with plant starch. For laforin and STBD1, an interaction with glycogen via the CBM20 domain was additionally demonstrated. In this work EDI3 could be associated for the first time with glycogen in vitro and with glycogen-associated proteins of the human skeletal and cardiac muscle. While no binding of EDI3 and glycogen could be detected in human liver, glucagon regulation was observed in primary mouse hepatocytes, potentially linking EDI3 to a role during gluconeogenesis. In skeletal muscle, EDI3 was associated with Type II skeletal muscle cells and the T-tubules, where it co-localizes with ryanodine receptor 1, suggesting a role of EDI3 in calcium signalling.2020-01-01T00:00:00ZStrukturbiologische Untersuchung und zelluläre Charakterisierung kovalent-allosterischer Akt-Inhibitoren
http://hdl.handle.net/2003/40093
Title: Strukturbiologische Untersuchung und zelluläre Charakterisierung kovalent-allosterischer Akt-Inhibitoren
Authors: Landel, Ina
Abstract: Aufgrund der Schlüsselposition der Proteinkinase Akt im PI3K/Akt-Signalweg resultiert aus genetischen Läsionen vorgeschalteter Proteine eine Überaktivierung der Kinase in verschiedenen Tumorerkrankungen. Zudem sind onkogene Mutationen der Akt-Kinase wie die E17K-Mutation bekannt, sodass die zielgerichtete Adressierung von Akt von großem Interesse in der medizinalchemischen Forschung ist. Dabei konnte das enorme Potential einer kovalent-allosterischen Modulation mit der Leitstruktur Borussertib durch dessen herausragende inhibitorische Potenz und Selektivität im Vergleich zu den in klinischen Studien untersuchten allosterischen und ATP-kompetitiven Akt-Inhibitoren bereits aufgelöst werden.
Mit den in dieser Arbeit etablierten Zellsystemen konnten weiterführend die gesteigerte anti-proliferative Aktivität von Borussertib im Vergleich zu den Referenzinhibitoren gezeigt und die selektive Inhibition der Akt-Kinase und der nachgeschalteten Signalkaskaden durch Borussertib intrazellulär bestätigt werden. Außerdem konnte die in vivo-Wirksamkeit von Borussertib in Kombination mit dem MEK-Inhibitor Trametinib in KRas-mutierten Patienten-abgeleiteten Xenograft-Modellen (PDX) nachgewiesen werden. Um eine ausreichende orale Bioverfügbarkeit zu erreichen, ist jedoch die weitere Optimierung von Borussertib im Rahmen des strukturbasierten Wirkstoffdesigns (SBDD) erforderlich. Dafür konnte in dieser Arbeit ein verlässliches Kristallisationssystem für die Akt1-Kinase etabliert und somit 35 Kristallstrukturen im Komplex mit einer Vielzahl von kovalent-allosterischen Inhibitoren gelöst werden. Die erhaltenen Komplexstrukturen bestätigen die Bindung in der allosterischen Interdomänentasche sowie die kovalente Adressierung der in der Aktivierungsschleife befindlichen Cysteine. Zudem konnte mit dem vertieften Verständnis der Struktur-Aktivitäts-Beziehungen (SAR) die Grundlage für die weitere Optimierung der CAAIs geliefert werden.
Um das SBDD auch zur Adressierung der onkogenen Akt1 E17K-Mutante zu ermöglichen, wurden zahlreiche Kristallisationsexperimente durchgeführt, welche letztendlich zur Identifizierung vielversprechender Bedingungen führten. Weiterhin konnten sowohl die biochemische als auch zelluläre Charakterisierung mit dem in dieser Arbeit generierten Ba/F3 Akt1 E17K-Zellsystem die Überlegenheit der CAAIs im Vergleich zu den klinischen Akt-Inhibitoren auch hinsichtlich der Adressierung der Akt1 E17K-Mutante auflösen.
Die validierten Methoden zur Etablierung eines Kristallisationssystems konnten außerdem auf die Rezeptortyrosinkinasen c-Kit- und PDGFR, welche prominente Zielproteine in gastrointestinalen Stromatumoren (GIST) darstellen, transferiert werden. Durch Reproduktion der beschriebenen Expressions-, Reinigungs- und Kristallisationsbedingungen konnten Kristalle des c-Kit Wildtyp-Proteins sowie vielversprechende Ansätze für PDGFR erhalten werden. Die Strukturanalyse der c-Kit-Kristalle lieferte dabei neue Ansätze zur Optimierung des Kristallisationskonstrukts.2020-01-01T00:00:00ZRole of WNT1-inducible signaling pathway protein-1 (WISP1) in liver injury
http://hdl.handle.net/2003/40035
Title: Role of WNT1-inducible signaling pathway protein-1 (WISP1) in liver injury
Authors: González Leiva, Daniela Fernanda
Abstract: Liver diseases are a global burden and a better understanding of factors controlling disease progression is required. In the last decade, much progress has been achieved in understanding the relevance of the extracellular matrix. Alterations in this dynamic structure can either facilitate or impair the repair of damaged liver tissue. Therefore, matricellular proteins of the CCN family, have emerged as new targets in liver pathophysiology. These highly conserved secreted proteins specifically interact with and signal through various extracellular partners, like integrins, which enable them to play crucial roles in various processes including development, wound healing and diseases such as cancer and fibrosis. We have discovered that WISP1 (Wnt1-inducible signaling pathway protein-1) also named CCN4, is induced upon CCl4-induced liver damage and may play an important role in the remodeling process of the extracellular matrix. Therefore, the aims were to identify the cell type that produces WISP1, to study its influence in cell migration and to use WISP1 KO mice to understand its role in the pathogenesis of liver fibrosis.
Isolation of individual liver cell types and quantification of WISP1 expression and secretion showed a higher mRNA expression and TGF-β-induced secretion of WISP1 in non-parenchymal cells, especially in stellate cells compared to hepatocytes. Furthermore, WISP1 facilitated the migration of isolated mouse hepatic stellate cells through collagen lattices, suggesting the interaction of WISP1 with one of the main components of the extracellular matrix and affecting its architecture. Additionally, gene expression analysis and Sirius Red staining showed differences in the development of CCl4-induced fibrosis between WISP1 wild type and knockout mice. Upregulation of collagen type I and α-SMA is reduced in WISP1 KO mice and less collagen deposition is also observed.
In conclusion, WISP1 is mainly expressed and secreted by stellate cells which may influence their migration upon liver injury and consequently, ameliorating the degree of liver fibrosis.2020-01-01T00:00:00ZIdentification and characterization of small molecules inducing cellular reactive oxygen species accumulation
http://hdl.handle.net/2003/40006
Title: Identification and characterization of small molecules inducing cellular reactive oxygen species accumulation
Authors: Wilke, Julian
Abstract: The human cell is constantly confronted with reactive oxygen species (ROS), which are oxygen-containing chemical intermediates readily reacting with different biomolecules. ROS can originate from exogenous sources, but are also produced within cells, e.g. as part of their metabolism. Depending on the concentration, ROS can either act as important signaling molecules or induce oxidative stress, thereby harming cellular components. Thus, cells utilize a multitude of antioxidant mechanisms to maintain redox homeostasis. As ROS play an important role in the development and propagation of cancer, modulation of cellular ROS levels, e.g. via small molecules, may have the potential to induce cancer-selective cytotoxicity.
In the course of this thesis, novel fast-acting small-molecule inducers of cellular ROS accumulation were identified by means of phenotypic screening. A counter-screen for glutathione-reactive compounds led to the discovery of 4,5-dihalo-2-methylpyridazin-3-ones and 2,3,4,5(6)-tetrachloro-6(5)-methylpyridines as potent depleters of cellular glutathione. Furthermore, an in-depth characterization of a 2,4-diaminopyrimidine-based compound class was conducted to identify the molecular target and elucidate the mode of action. Structure-activity relationship studies improved both potency and water solubility of the original hit compound and eliminated bioactivities unrelated to its ROS-inducing properties. The optimized compound DP68 was found to strongly elevate cellular superoxide levels without affecting hydrogen peroxide concentrations, indicating an interference with superoxide dismutation. However, an inhibition of the enzymatic activity of superoxide dismutases by DP68 was not observed. Target identification studies of DP68 were conducted, using affinity-based chemical proteomics and different profiling techniques to investigate the compound’s influence on cellular morphology, thermal stability of proteins and the (phospho)proteome. Thereby, DP68 was identified as novel antagonist of the σ1 receptor, which however does not mediate ROS induction. Furthermore, DP68 was found to interact with HEAT repeat-containing proteins, including phosphatidylinositol 3-kinase-related kinases. In addition, DP68 induced a phosphorylation of superoxide dismutase 1, which may influence its cellular localization. DP68 induced cytotoxicity in cancer cell lines and non-malignant primary cells, which may depend on its superoxide-inducing properties.; Die menschliche Zelle gerät dauerhaft mit reaktiven Sauerstoffspezies (ROS) in Kontakt. Dabei handelt es sich um sauerstoffhaltige chemische Intermediate, die spontan mit verschiedensten Biomolekülen reagieren können. ROS können sowohl aus exogenen Quellen stammen, aber auch in Zellen selbst, z.B. als Nebenprodukt ihres Metabolismus, entstehen. Abhängig von ihrer Konzentration können ROS sowohl als Signalmoleküle fungieren, oder oxidativen Stress auslösen und zelluläre Komponenten schädigen. Aus diesem Grund verfügen Zellen zur Erhaltung ihrer Redoxhomöostase über eine Vielzahl antioxidativer Mechanismen. Da ROS auch bei der Entstehung und Ausbreitung von Tumoren wichtige Rollen spielen, könnte ihre Modulation, zum Beispiel durch niedermolekulare Substanzen, das Potential zur Induktion krebsselektiver Zytotoxizität innehaben.
Im Rahmen dieser Doktorarbeit wurden mithilfe eines phänotypischen Screens neuartige schnellwirksame niedermolekulare Substanzen identifiziert, die die zelluläre ROS-Konzentration erhöhen. Ein weiterer Screen zur Detektion glutathionreaktiver Substanzen führte zur Identifizierung von 4,5-Dihalogen-2-methylpyridazin-3-onen und 2,3,4,5(6)-Tetrachlor-6(5)-methylpyridinen, die eine zelluläre Glutathiondepletion induzieren. Des Weiteren wurden ausführliche Studien einer 2,4-Diaminpyrimidin-basierten Substanzklasse durchgeführt, um das Zielmolekül zu identifizieren und den Wirkmechanismus aufzuklären. Die Untersuchung der Struktur-Aktivitätsbeziehung führte zur Verbesserung der Potenz sowie der Wasserlöslichkeit und zur Eliminierung von Bioaktivitäten, die nicht mit der ROS-induzierenden Eigenschaft in Verbindung stehen. Untersuchungen der optimierten Substanz DP68, zeigte eine starke Erhöhung zellulärer Superoxidlevel ohne Änderung der Wasserstoffperoxidkonzentration, welches darauf hinweist, dass die Substanz mit der Superoxiddismutation interferieren könnte. Eine Inhibition der enzymatischen Aktivität von Superoxiddismutasen durch DP68 wurde jedoch nicht festgestellt. Zur Identifizierung der molekularen Zielstruktur von DP68 wurden Studien mittels affinitätsbasierter chemischer Proteomik und verschiedener Profilingtechniken, die den Einfluss der Substanz auf die zelluläre Morphologie, die thermische Stabilität von Proteinen und das (Phospho)proteom untersuchen, durchgeführt. Dabei wurde DP68 als neuartiger Antagonist des σ1-Rezeptors identifiziert, jedoch steht diese Interaktion nicht mit der ROS-Induktion in Verbindung. Desweiteren interagiert DP68 mit verschiedenen Proteinen, die „HEAT repeat“-Strukturmotive enthalten, z.B. mit Kinasen der „Phosphatidylinositol 3-kinase-related kinase“-Familie. Zusätzlich wurde die eine Phosphorylierung der Superoxiddismutase 1 nach Behandlung mit DP68 festgestellt, welches einen Einfluss auf dessen zelluläre Lokalisation haben könnte. DP68 induzierte Zytotoxizität sowohl in Krebszelllinien, als auch in nicht-malignen Primärzellen, welche auf seiner superoxidinduzierenden Eigenschaft beruhen könnte.2020-01-01T00:00:00ZCollective information processing in synthetic morphogenic liposomes and hybrid cell-computer wound healing with optogenetically emulated signaling
http://hdl.handle.net/2003/39980
Title: Collective information processing in synthetic morphogenic liposomes and hybrid cell-computer wound healing with optogenetically emulated signaling
Authors: Scocozza, Bruno
Abstract: Throughout the different scales at which living organisms operate, it is the collective behaviour
between the entities that compose them that determines their identity and function.
Biomolecules, cells and whole organisms interact to process information and generate
organization at the subcellular, tissue and ecosystem scales.
In the first part of this thesis, we investigated fundamental principles of cellular morphogenesis
in response to external cues in a Synthetic Morphogenic Membrane System (SynMMS).
SynMMS was composed of an encapsulated dynamic microtubule (MT) aster, together with a
light-inducible signal transduction system in cell-sized liposomes. We show that light-activated
signaling induces MT growth through activation of an activity gradient of the MT-regulator
stathmin. SynMMSs are thus capable of responding to local external light cues with directed
morphological changes, as if originating from an extracellular morphogen. Morphometric
analysis methods enabled us to uncover feedbacks within and among the signaling and
cytoskeletal subsystems, which are mediated by the deformable membrane. We found that bidirectional
interactions between the signaling and MT-aster system underlie the creation of
self-organized morphologies as well as their reorganization in the direction of external light
cues.
In the second part, we develop a novel method to investigate how at a higher scale, the coupling
of such intracellular processing networks leads to functional collective behaviour. We develop
a hybrid cell-computer experimental interface to create hybrid wound healing assays, in which
living cells are recruited to a virtual wound while undergoing virtual paracrine signaling. The
migration state of cells is measured in real time from which in silico cell-to-cell communication
is computed and translated into optogenetic actuation that induces cell migration, emulating
extracellular signaling cues. The hybrid assay phenocopies features of in vivo neutrophil
swarming towards an injury, demonstrating that a paracrine signaling relay allows expanding
the recruitment region. Thus, this method allows to create hybrid in vitro-in silico dynamics
where the influence of intercellular communication parameters can be quantitatively
investigated.
Altogether, investigation of collective behaviour in synthetic proto-cells uncovered how MTsignaling
interactions determine cellular morphogenesis, while hybrid cell-computer interfaces establish a new method for quantitative interrogation of cellular communication during tissue
morphogenesis.2020-01-01T00:00:00ZRegulation of natural killer cell cytotoxic pathways during serial killing activity
http://hdl.handle.net/2003/39816
Title: Regulation of natural killer cell cytotoxic pathways during serial killing activity
Authors: Prager, Isabel
Abstract: Natural Killer (NK) cells act as the front line of the body´s defense in the immune system and they are key players in efficiently recognizing and eliminating virus-infected and tumor cells. Individual NK cells can eliminate multiple target cells in a sequential manner during a process named serial killing. There are two major pathways NK cells use to induce apoptosis in their target cells: By releasing the content of cytotoxic granules containing the serine protease granzyme B (GrzB) and the pore-forming protein perforin or by the engagement of death receptors, which initiates caspase cascades via Caspase-8 (Casp8). The contribution of both cell death processes to NK cell cytotoxicity and serial killing remains poorly understood. Therefore, investigating the interplay between these pathways in more detail is important for a better understanding of NK cell cytotoxicity. To visualize the GrzB and death receptor-mediated target cell death in a time-dependent manner, we used fluorescent localization reporters that enabled us to simultaneously measure the activities of GrzB and of Casp8 in target cells upon contact with NK cells by life cell imaging. In this study we observed that NK cells kill their initial targets via the fast GrzB-induced pathway and switch to a slow death receptor-mediated killing for the final target. During the target cell contact NK cells lost GrzB and perforin, whereas the expression of CD95L, a main death cell ligand, was increased on the NK cell surface. The reduction of the lytic granules can be efficiently restored by the stimulation with different cytokines such as IL- 15, IL-2 or IL-21. Perforin deficient NK cells or ILC3 cells were unable to perform GrzB-mediated killing and no serial killing could be mediated without this pathway. In contrast, the absence of death receptor CD95 on the target cell and/or the absence of death receptor ligands on the NK cell had no direct influence on the GrzB-mediated serial killing. This demonstrates that the use of GrzB vs. death receptor-mediated target cell killing is differentially regulated during the serial killing activity of NK cells. Taken together, we observed a rapid target cell death which was induced by GrzB and originated from early established NK : target contacts. In contrast, cell death mediated by Casp-8 was a result of later target cell engagements and took much longer from NK : target cell contact to target cell death.2020-01-01T00:00:00ZAlterations of the glyoxylate metabolism in hepatic steatosis – a risk factor for hyperoxaluria
http://hdl.handle.net/2003/39769
Title: Alterations of the glyoxylate metabolism in hepatic steatosis – a risk factor for hyperoxaluria
Authors: Jashari, Adelina
Abstract: Non-alcoholic fatty liver disease (NAFLD) comprises a wide spectrum of liver diseases and is the leading cause of liver diseases worldwide. Features of the metabolic syndrome and extrahepatic diseases, like cardiovascular diseases and kidney stone disease, strongly associate with NAFLD. A previously identified steatosis-associated downregulation of the alanine-glyoxylate aminotransferase (AGXT) was suggested to be one molecular link explaining the connection of NAFLD with the development of calcium oxalate stones. AGXT is responsible for the hepatic glyoxylate detoxification and its deficiency results in primary hyperoxaluria type 1 (PH1), which is a hereditary disorder leading to the formation of calcium oxalate kidney stones. Furthermore, the AGXT downregulation in steatotic conditions was proposed to be influenced by DNA methylation since the AGXT promoter is hypermethylated in steatosis. In the course of this thesis, further alterations of the glyoxylate metabolism were revealed in the ob/ob and Western diet (WD) mouse models of NAFLD contributing to a better understanding of the steatosis-associated modifications. These alterations indicating an increased hepatic oxalate production in steatotic conditions were confirmed by elevated oxalate excretion of steatotic hepatocytes from WD-fed and ob/ob mice upon hydroxyproline exposure. Furthermore, the increased oxalate production in the fatty liver was validated in vivo by elevated oxalate levels in the plasma from the hepatic vein of WD-fed mice. Additionally, oxalate levels in the plasma and urine of ob/ob mice were increased to a higher extent by dietary hydroxyproline compared to ob/+ mice, showing an enhanced susceptibility towards hydroxyproline also in vivo. The selective sensitivity of steatotic hepatocytes to hydroxyproline contrasted with that of primary hepatocytes from Agxt knock-out (Agxt-/-) mice, which excreted more oxalate when exposed to all oxalate precursors than hepatocytes from wild type control mice. This suggested, that only mitochondrial glyoxylate detoxification is compromised in hepatic steatosis. Rescuing the Agxt expression in the hepatocytes of ob/ob mice by AAV-mediated gene transfer was able to reduce oxalate production from hydroxyproline. Moreover, inhibition of hydroxyproline catabolism normalised the oxalate excretion from ob/ob hepatocytes after consumption of hydroxyproline. These results provided clear evidence that the downregulation of Agxt is, at least partially, responsible for the steatosis-accompanied increased hepatic oxalate production in NAFLD mouse models. This supported the hypothesis of the steatosis-associated downregulation of Agxt being a molecular link explaining the strong association between NALFD and kidney stone disease. The translational relevance of these findings was studied in a cohort of overweight and obese children and adolescents with biopsy proven NAFLD and corresponding 24 h urine samples. The steatosis percentage positively correlated with the amount of urinarily excreted oxalate, showing the relevance of steatosis induced hyperoxaluria in human NAFLD. In order to further understand the deregulation of Agxt expression in steatosis and the dependency to DNA methylation, its transcriptional upregulation in response to glucagon was studied in ob/ob and 6 weeks WD-fed mice. When treated with glucagon, an increase of the Agxt mRNA expression was missing in ob/ob mice but not in 6 weeks WD-fed mice, most probably due to the higher hypermethylation of the Agxt promoter in ob/ob mice. This supported the importance of the Agxt promoter hypermethylation regarding the regulation of the Agxt gene expression. These results were confirmed in vitro in glucagon stimulated primary hepatocytes from the two different NAFLD mouse models and in an in vitro steatosis model. Furthermore, the phosphorylation of Creb was lost earlier in hepatocytes from ob/ob, 6 weeks WD-fed mice and in an in vitro steatosis model. All in all, these findings support the thesis that steatosis associated AGXT promoter hypermethylation might repress the AGXT gene expression leading to an impaired response towards glucagon.2020-01-01T00:00:00ZLight-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization
http://hdl.handle.net/2003/39300
Title: Light-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization
Authors: Palei, Shubhendu; Buchmuller, Benjamin; Wolffgramm, Jan; Muñoz-Lopez, Álvaro; Jung, Sascha; Czodrowski, Paul; Summerer, Daniel
Abstract: Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-l-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.2020-04-14T00:00:00ZClick-mediated enrichment of specific genomic loci
http://hdl.handle.net/2003/39245
Title: Click-mediated enrichment of specific genomic loci
Authors: Witte, Anna
Abstract: In all organisms, the genetic information of cells is stored in the nucleotide sequence of deoxyribonucleic acid (DNA). The human organism consists of more than 200 different somatic cell types with the same genetic information (genotype). Even though, they drastically differ in their morphology and function (phenotype), which is related to different gene expression levels. Gene expression is controlled by macromolecular interactions and epigenetic modifications on chromatin that are highly locus-specific and drive functional aspects of each locus. Even though, the compositions of macromolecules and modifications on many chromosome loci remain poorly understood, in part due to the lack of locus-specific chromatin purification methods that would allow for targeted, discovery-oriented analyses.
In this work, the first enrichment method based on bio-orthogonal conjugation (“click-chemistry) with encoded programmable DNA binding domains (transcription-activator like effectors – TALEs) for purification of user-defined genomic loci was established. This click-mediated enrichment provides complementary potential compared to the existing enzymatic biotinylation strategies used in chromatin enrichment methods in the view of site-specificity and proteome-wide background. This method will enable correlations of local chromatin states with phenotypes as the key to a deeper understanding of the regulation landscape of the eukaryotic genome. As a first outlook experiment, we extended our approach to fusion constructs of specific TALE proteins and ten-eleven translocation (TET) dioxygenases for epigenetic editing in vivo. TETs catalyze the oxidation of 5-methylcytosine (5mC) to the oxidized derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). In combination with the click-mediated enrichment and proteomics analysis, this will enable studying how local epigenetic changes modulate the local chromatin landscape in vivo as basis for alterations in gene expression.2020-01-01T00:00:00ZDirected evolution of lysine deacetylases
http://hdl.handle.net/2003/39244
Title: Directed evolution of lysine deacetylases
Authors: Spinck, Martin
Abstract: Every cell in the human body contains the exact same genetic information in their
nucleus but despite this cell function, structure and content can differs significantly.
The reason for this is found in the epigenetic information, a complex code of chemical
modification on the chromatin proteins and DNA which determines cellular identity.
This code recently grew in complexity with the discovery of lysine acylation on histone
proteins. Lysine acylation is related to lysine acetylation and spans a wide range of
acyl-Coa derived modifcations like lysine propionylation, butyrylation, or crotonylation
up to long chained myristoylation. All these modifications are installed and removed by
a relatively small set of substrate promiscuous lysine acetyltransferases and
deacetylases, respectively. The large extend of substrate promiscuity has hindered our
understanding of acylation as part of the epigenetic code so far.
Here I present a directed evolution-based approach to alter deacylation selectivity of
lysine deacetylases allowing for the manipulate of cellular acylation patterns and I
developed novel methodology to rapidly measure deacylation activity. Both methods
are based on genetic code expansion, a method to genetically encode unnatural amino
acids in place of an amber stop codon into proteins, including various lysine acylations.
I found that by acylation of the catalytic lysine of Orotidine 5'-phosphate decarboxylase
(Ura3) its activity becomes dependent on deacylation and allows for selection of E.coli
cells. Through the use of acylated firefly luciferase a highly sensitive KDAC deacylation
assay was established.
The Ura3 selection system was able to select the E.coli Sirtuin CobB, and the human
Sirtuins 1, 2, 3, 6 and 7 as well as the human histone deacetylase HDAC8. Selection
of 40 million CobB mutants produced a wide range of acyl-selective mutants, as well
as mutants exhibiting deallylation and boc- deprotection activity. Particularly interesting
was the CobBac2 mutant, which lost all decrotonylation activity but maintained all other
deacylation activities. The crystal structure of CobBac2 revealed that crotonyl binding
stabilized a novel conformation of the cofactor binding loop. Addition of NAD+ caused
the reaction to stop at intermediate 3, which could not be hydrolyzed due to the
positioning of the cofactor binding loop. Expression of CobBac2 in mammalian cells
removed all acylation except crotonylation, showing its potential to alter the epigenetic
code. The same selection procedure was used to identify acetyl selective Sirt1 mutants
and to isolate Sirt6 mutants with increased deacetylation activity.
The acylated firefly luciferase was further developed into an assay for drug discovery.
In cooperation with the COMAS we identified novel scaffolds for Sirt1 inhibition, which
is comparable in potency to Ex527 but more selective for Sirt1. Screening of activators
could not identify potent chemical activators, but surprisingly we found that the native
C-terminal lamin A peptide strongly activates Sirt1 decrotonylation.
In the future acyl-selective deacetylases will make an important contribution to a better
the understanding of acylation in various biological processes such as aging, cell
differentiation and metabolism.2020-01-01T00:00:00ZBiochemische, kinetische und zelluläre Charakterisierung kovalent-allosterischer Akt-Inhibitoren
http://hdl.handle.net/2003/39238
Title: Biochemische, kinetische und zelluläre Charakterisierung kovalent-allosterischer Akt-Inhibitoren
Authors: Weisner, Jörn
Abstract: Die Proteinkinase B (auch: Akt) spielt eine essenzielle Rolle in einer Vielzahl zellulärer Signaltransduktionskaskaden, welche grundlegende Prozesse wie Proliferation, Überleben und Apoptose regulieren. Eine Fehlregulation von Akt ist mit diversen kardiovaskulären, neurodegenerativen sowie onkologischen Krankheitsbildern assoziiert. Häufig sind genetische Läsionen in den vorgeschalteten Proteinen wie PI3K und PTEN ursächlich für derartige Erkrankungen, aber auch Alterationen in membranständigen Rezeptor-Tyrosinkinasen wie EGFR und c-Kit können pathologische Änderungen in der Akt-vermittelten Signalweiterleitung bewirken. Direkte Läsionen in Akt, wie bspw. die aktivierende somatische Mutation E17K oder eine Amplifikation und Überexpression von Akt, treten weniger häufig auf, können jedoch ebenfalls als treibender Bestandteil in der Pathogenese involviert sein. Eine pharmakologische Inhibition von Akt kommt daher als vielseitige Strategie für die therapeutische Behandlung diverser Erkrankungen in Frage. Neben den klassischen ATP-kompetitiven Inhibitoren, welche zumeist unzureichende Selektivitätsprofile aufweisen und signifikante Nebenwirkungen induzieren, existieren für Akt auch allosterische Liganden, welche an der Grenzfläche zwischen regulatorischer PH-Domäne und katalytischer Kinase-Domäne binden. Dieser einzigartige Bindungsmodus gewährt eine ausgezeichnete Selektivität für das Zielprotein verbunden mit einer Minimierung der möglichen off-target-vermittelten Toxizitäten.
In der vorliegenden Arbeit wird das rationale Design sowie die Charakterisierung von kovalent-allosterischen Akt-Inhibitoren in biochemischen und zellulären Systemen beschrieben. Basierend auf Komplexkristallstrukturen mit reversibel bindenden Liganden wurden in computergestützten Modellierungsansätzen initial neue Moleküle, welche zusätzlich mit einem Cystein-reaktiven Michael-Akzeptor dekoriert sind, entworfen. Mithilfe von massenspektrometrischen und Aktivitäts-basierten Analysen konnte für eine fokussierte Substanzbibliothek auf Basis des 1,6-Naphthyridinon-Grundgerüsts ein kovalenter Bindungsmechanismus sowie eine potente Inhibition von Akt1 in vitro nachgewiesen werden. In weiterführenden strukturbiologischen Untersuchungen konnten für vier der dargestellten Verbindungen Komplexkristallstrukturen mit Akt1 gelöst und hierdurch die Besetzung der allosterischen Bindetasche in Akt1 sowie die irreversible Bindung zu Cys296 bzw. Cys310 nachgewiesen werden. Unter Verwendung von zellulären, Krebs-relevanten Modellsystemen konnte die anti-proliferative Wirksamkeit dieser neuartigen Substanzklasse abgebildet werden, welche anhand von Western Blot-Studien auf die on-target-Inhibition von Akt zurückgeführt werden konnte. In Folge von in vitro pharmakokinetischen Untersuchungen konnte mit Verbindung 3a (Borussertib) eine Leitstruktur identifiziert werden, deren vielversprechenden Aktivitäts-, Selektivitäts- und PK-Profile eine anschließende Evaluierung in in vivo-Modellen erlaubten. Trotz einer unzureichenden oralen Bioverfügbarkeit sowie einer moderaten MTD von 20 mg/kg bei intraperitonealer Applikation konnte die in vivo-Wirksamkeit der Leitstruktur in Patient-abgeleiteten Xenograft-Modellen des Kolorektal- sowie Pankreaskarzinoms in Kombination mit dem MEK-Inhibitor Trametinib gezeigt werden. Diese Eigenschaften verdeutlichen das Potential der kovalent-allosterischen Akt-Inhibition und bilden zeitgleich das Rational für die Weiterentwicklung und Optimierung der Leitstruktur zum klinischen Kandidaten.
Ein weiteres Projekt beinhaltete die Identifikation und Charakterisierung von Modulatoren des Keap1/Nrf2-Signalwegs, welcher zytoprotektive Funktionen gegenüber oxidativem Stress und Entzündungsprozessen, die in der Entstehung von kardiovaskulären und neurodegenerativen Krankheitsbildern beteiligt sind, vermittelt. Zusätzlich kann eine Fehlregulation von Nrf2 für die Tumorentstehung und progression förderlich sein. Unter Verwendung eines Fluorophor-markierten, Nrf2-mimikrierenden Sondenpeptids und der rekombinanten Kelch-Domäne von Keap1, welche die Protein-Protein-Interaktion mit Nrf2 vermittelt, wurde ein Hochdurchsatz-kompatibler, Fluoreszenzpolarisations-basierter Assay entwickelt. Mithilfe dieses Assays und der Screening-Plattform RASPELD wurde die ca. 30000 Substanzen-umfassende in-house Bibliothek nach Modulatoren der Keap1/Nrf2-PPI durchmustert. Zwar konnte keins der identifizierten Hit-Moleküle in Folgestudien als Inhibitor validiert werden, der entwickelte Assay erweitert dennoch das Portfolio der Screening-Plattform RASPELD und fungiert dabei als Grundlage für zukünftige Screening-Kampagnen auch hinsichtlich der Identifikation von möglichen Stabilisatoren der Keap1/Nrf2-Interaktion.
Desweiteren wurden im Rahmen der vorliegenden Arbeit Elektronenspinresonanz-Spektroskopie-basierte Studien an der p38α MAPK hinsichtlich der konformationellen Plastizität und Aktivierungsschleifendynamik durchgeführt. Hierfür wurde ein heterolog exprimiertes und positionsspezifisch mit dem spin label MTSSL markiertes p38α-Konstrukt eingesetzt und in An- und Abwesenheit von Typ I- und Typ II-Inhibitoren charakterisiert. Die rotatorische Mobilität des eingebrachten Spinmarkers ermöglichte hierbei die Quantifizierung von konformationellen Zuständen in p38α in Abhängigkeit der Ligandenbindung. Mithilfe dieser Methodik konnten zwei separate Zustände der Aktivierungsschleife identifiziert werden, welche der DFG-in- bzw. der DFG-out-Konformation zugeordnet werden konnten und im apo-Zustand im Verhältnis 9:1 vorlagen. Anders als in vorhandenen Komplexkristallstrukturen konnte für keinen der getesteten Typ I- bzw. Typ II-Inhibitoren eine vollständige Verschiebung des Konformations-gleichgewichts zugunsten eines einzelnen Zustands beobachtet werden. Stattdessen waren unabhängig vom gebundenen Liganden beide Konformationen koexistent. Temperaturabhängige Messungen erlaubten die Evaluierung des Konformationsübergangs von DFG-in nach DFG-out auf thermodynamischer Ebene. Im apo-Zustand sowie in der Typ I-Inhibitor-gebundenen Form von p38α war die Gibbs-Energie ΔG positiv, d. h. der Konformationsübergang ist energetisch ungünstig. Für die Typ II-stabilisierte Konformation wurden im Vergleich geringere Reaktionsenthalpien und -entropien ermittelt, was wiederum für eine bevorzugte Adaption der DFG-out-Konformation spricht. Neben der thermodynamischen Differenzierung zwischen Typ I- und Typ II-Inhibitoren bilden die hier präsentierten Ergebnisse auch die Grundlage für detaillierte Folgestudien wie bspw. Abstands-basierte Multilaterationsansätze. Diese ermöglichen eine dreidimensionale Kartierung von flexiblen und dynamischen Strukturelementen in Proteinen, wie bspw. der Aktivierungsschleife in Proteinkinasen, die in Kristallstrukturen häufig unzureichend aufgelöst sind.2020-01-01T00:00:00ZAnwendung der hochaufgelösten bildgebenden MALDI-MS zur Aufklärung der räumlichen Verteilung des Acetaminophens und seiner Biotransformationsprodukte sowie von Taurocholaten in der Leber von Mäusen in Folge einer Acetaminophen Intoxikation
http://hdl.handle.net/2003/39237
Title: Anwendung der hochaufgelösten bildgebenden MALDI-MS zur Aufklärung der räumlichen Verteilung des Acetaminophens und seiner Biotransformationsprodukte sowie von Taurocholaten in der Leber von Mäusen in Folge einer Acetaminophen Intoxikation
Authors: Sezgin, Selahaddin
Abstract: Überdosierungen an APAP führen zu hepatotoxischen Auswirkungen in der Leber von Säugetieren. Die Hepatotoxizität ist u.a. eine Folge der metabolischen Aktivierung des Acetaminophens durch CYP. Dabei treten pericentrale Schadensmuster in den Leberläppchen auf. Es fehlten bisher Informationen über die räumlich-zeitliche Verteilung vom APAP und seinen Biotransformationsprodukten in der Leber bis zur Veröffentlichung von Teilen dieser vorliegenden Arbeit. Um die Fragestellung zu beantworten, wurde eine Methode basierend auf der leistungsstarken HR-MALDI-MSI Technik entwickelt. Dadurch konnte die Verteilung vom APAP, seinen Hauptmetaboliten APAP-GLC und APAP-SUL und seinem Addukt APAP-GSH in der Leber von C57BL/6 Mäusen, denen APAP intraperitoneal verabreicht wurde und deren Leber sowohl zeit- als auch dosisabhängig beprobt wurden, detektiert werden. Zuvor wurden intensive Anstrengungen unternommen, um das toxische Intermediat NAPQI im Lebergewebe durch MALDI-MSI zu erfassen. Ziel war es, Informationen über seinen Bildungs- und Wirkungsort im Lebergewebe zu gewinnen. Hervorzuheben ist dabei ein Versuchsansatz bei dem H2S zur Derivatisierung von NAPQI eingesetzt wurde. Diese innovative Methode könnte zudem das Portfolio an Derivatisierungsmöglichkeiten für MALDI-MSI Experimente erweitern. Allerdings konnte das NAPQI weder direkt noch über seine artifiziellen Derivate im Lebergewebe detektiert werden, was vermutlich auf einen Mangel an nicht-abreagiertem NAPQI im Lebergewebe zurückzuführen ist. Die durch Gewebehistologie unterstützte Superpositionierung von Ionendichtebildern unter Einbeziehung der Eigenschaften verschiedener Imaging Software ermöglichte es, präferentielle Verteilungen der Analyten bezüglich der metabolischen Zonen zu erfassen. Während für APAP, APAP-SUL und APAP-GLC keine zonale Verteilung festgestellt werden konnte, konnte eine präferentielle Bildung des APAP-GSHs sowie ein schwerpunktmäßiger Abbau des GSHs in der pericentralen Zone verortet werden. Der Abbau an GSH war aber nicht ausschließlich auf die pericentralen Zonen beschränkt, sondern konnte auch für die periportalen Zonen demonstriert werden. 120 min nach der APAP Injektion konnte ein Influx an APAP-GSH und an Taurocholaten von den Gallengefäßen in die pericentrale Zone und von dort ins Blut beobachtet werden. Dieser massive Eintrag von Gallensäuren beweist, dass sich ein „sekundäres“ Toxizitätsevent ereignet, welches vormals durch die Aktivierung des APAPs in der Leber gestartet wurde.; Acetaminophen induces hepatotoxicity in the liver of mammal organisms, after the uptake of an overdose. It is well reported, that CYP mediated activation of APAP is thereby causing a pericentral damage pattern in the liver lobules. However, no direct spatio-temporal information about the distribution pattern of APAP and its biotransformation products in the liver were available until the publication of parts of this work. Herein, the development of an analytical method based on of the sophisticated HR-MALDI-MSI technique is reported. The aim was to detect the distribution of APAP, its main metabolites APAP-GLC and APAP-SUL and adduct APAPGSH, known to be formed during detoxification of the toxic intermediate metabolite NAPQI by GSH, in the liver of C57BL/6 mice, which were treated intraperitoneally with APAP and whose livers were collected in a time-and dose-dependent manner. Previously, intensive efforts were put in the analytical investigation of the toxic intermediate NAPQI from liver tissue by MALDIMSI since its detection would have revealed valuable information about its sites of formation and impact. For this purpose, an innovative derivatization approach with H2S gas was applied, which in general, could also enrich the portfolio of derivatization methodologies used for MALDIMSI experiments. However, the detection of NAPQI and its on-tissue derivatization attempts failed, presumably due to the lack of unreacted NAPQI in the liver. Histology-directed superposition of ion density images in consideration of special features of different imaging software made it possible to determine the differences in the distribution pattern of the analytes. While no specific zonation could be detected for APAP, APAP-SUL and APAP-GLC, a preferential formation of APAP-GSH and depletion of GSH in the pericentral zones of the lobule were revealed. However, the depletion of GSH was not restricted to pericentral zones but was also demonstrated for the periportal areas. 120 min after APAP injection a series of drastic events happened: APAP-GSH as well as taurocholates entered from the bile vessels into the pericentral zone and from there leaked into the systemic blood circulation. The massive leakage of bile acids into the hepatocytes is providing the evidence of a “secondary” toxicity event initiated by the activation of APAP in the liver.2019-01-01T00:00:00Z