Max-Planck-Institut für molekulare Physiologie

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Design and synthesis of photoactivatable myristic acid analogues for UNC119 cargo interactions and target identification of autophagy inhibitors
(2019) Kaiser, Nadine; Waldmann, Herbert; Summerer, Daniel
UNC119 is a chaperone protein, which regulates the trafficking of myristoylated proteins between cellular membranes. Even though these chaperones were extensively studied in the past two decades, only few of their interaction partners are known to date. In the course of this thesis three different photoactivatable myristic acid analogues (Pacman 1-3) were designed and synthesized to study this protein-myristate interaction. Exclusively Pacman-3-based probes covalently labeled UNC119 proteins. The photocrosslinking site of Pacman-3 probes within the hydrophobic pocket of UNC119 was characterized. The myristate analogues binding mode is similar to the native myristate moiety. Substrate recognition of Pacman-3 by N-myristoyl transferase (NMT) enzymes from different organisms was investigated. Pacman-3 was not incorporated into a peptide substrate by human NMT. Remarkably, NMT derived from the protozoan parasite Leishmania and Trypanosoma recognized Pacman 3 as a substrate. Conclusively, Pacman-3 can serve as a valuable tool to investigate the role of UNC119 protein in these parasites and to identify novel myristoyl-interaction partners. The second project of this thesis focused on autophagy. Autophagy is an essential biological process for the regulation of cellular homeostasis and energy supply. Modulators of autophagy can provide insight into the regulation of autophagic flux and unravel its role in various diseases. In order to identify novel autophagy modulators, a phenotypic screen was performed. Three structurally different potential inhibitors of autophagy were investigated in detail. The first compound was a representative of the oxazolidinone-scaffold, namely Autoxain. Initial experimental results indicated a similar mode of action as the antibiotics linezolid and tedizolid, which exert their antibacterial activity by inhibition of protein biosynthesis via the ribosome. The second small molecule was Autoquin, which prevents autophagosome-lysosome fusion. An affinity enrichment experiment suggested a similar mode of action to the known autophagy inhibitors salinomycin and ironomycin, which sequester iron in the lysosome. Autophagy plays a crucial role in maintenance of cellular iron homeostasis and novel tool compounds, which could shed light on this biological process are highly relevant. The autophagy inhibitor Authipyrin interferes with mitochondrial respiration by direct inhibition of complex I. Complex I activity is required for regular autophagic flux. Furthermore, mitophagy is frequently dysregulated in patients suffering from Parkinson’s disease. However, the exact mechanism underlying this interplay remains to be elucidated.
Spatial unification of coupling interactions between EGFR and PTPs establishes a growth factor sensing network
(2018) Stanoev, Angel; Koseka, Aneta; Bastiaens, Phillippe
Cells continuously sense and respond to stimuli from the non-stationary environment. For this, they optimise processing of the perceived signals while maintaining continuous responsiveness. Cell surface receptors, such as the receptor tyrosine kinases, comprise the first layer of sensing. They translate the extracellular signal into internal activity using the protein interaction networks in which they are embedded. The proto-oncogenic epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose sensitivity to epidermal growth factor (EGF) and signalling duration determines cellular behaviour. In the canonical view, signal processing occurs through the ligand-induced dimer formation mechanism and subsequent trans-phosphorylation. However, it has been also established recently that signal amplification via the unliganded EGFR monomers through an autocatalytic mechanism enhances the phosphorylation response of EGFR. In this thesis, I demonstrate how autocatalytic phosphorylation of EGFR in concert with the coupling interactions with the protein tyrosine phosphatases (PTPs) shape the response dynamics of EGFR. Single cell dose-response analysis revealed that a toggle switch between autocatalytically activated monomeric EGFR and the tumour suppressor PTPRG at the plasma membrane (PM) shapes the sensitivity of EGFR to EGF dose. As the system exhibits switch-like activation due to the bistable regime of operation, irreversible activation occurs as an adversary side effect. To ensure continuous growth factor sensing, the system is positioned outside of the bistable regime by the PM-localised PTPRJ, which negatively regulates EGFR phosphorylation. On the other hand, a spatially-distributed negative feedback with the ER-bound PTPN2 that is established by vesicular trafficking resets the phosphorylation state of monomeric EGFR on the plasma membrane. The distinct recycling route of the unliganded receptor, as opposed to the unidirectional degradation route towards the perinuclear area of the liganded receptor, enables it to repopulate the plasma membrane and thus maintain sensitivity to upcoming stimuli. In this manner, the coupling interactions between EGFR and the PTPs on different membranes are spatially unified in a network that enables sensing of time-varying EGF signals. The signal processing capabilities of this network are optimised by the system organisation, as its parameters are poised at the criticality point, just outside the bistable regime of operation. In this region, the EGFR response is characterised by prolonged but reversible phosphorylation, enabling the cell to maintain a balance between preserving a transient memory of previous EGF stimulations, while still remaining responsive to upcoming stimuli.
Distinct focal adhesion protein modules control the adhesion site segregation and cell migration behavior
(2017-11) Imtiaz, Sarah; Philippe, Philippe; Dehmelt, Leif
Cell adhesion and migration require a tightly regulated organization of cytoskeleton and cell-matrix adhesion sites which are large protein complexes consisted of multiple components. To address the question how the core focal adhesion (FA) proteins mediate the adhesion segregation process to form fibrillar adhesions (FB) and how they regulate cell motility, a high-content imaging screen was employed using combinatorial RNAi for 10 FA proteins. This revealed distinct FA proteins directly influence the segregation of adhesion sites, suggesting the modular assembly and function of adhesion sites. We identified three distinct modules: signaling, actin-regulatory and adhesion building-segregating module. Signaling proteins such as FAK and p130CAS as well as actin-regulatory proteins (VASP, α-actinin-1, zyxin) control the localization of tensin-1 in FA, which is the hallmark of FB. Structural proteins (kindlin-2, ILK and talin-1) modulate the translocation of tensin-1 from focal adhesion to FB. Notably the same set of proteins, were associated with specific modes of migration. Signaling and actin-regulatory module is linked to enhance cell migration (amoeboid and lamellipodial modes), whereas the adhesion building-segregating module is related to impaired migration (confined mode). Taken together, this study shows that FA are composed of different functional modules that distinctly control different stages of the adhesion transformation process and the cell migration behavior.
Entwicklung von Inhibitoren für Lipoproteintransporterproteine, Hh-Coaktivatoren und einer γ-selektiven C-Ferrier-Umlagerung
(2018) Hofer, Walter; Waldmann, Herbert; Summerer, Daniel
Krebs zeichnet sich durch eine Fehlregulierung entscheidender Prozesse in der Zelle aus. Eine der prominentesten Vertreter, die zur Deregulierung als Onkogene für wichtige Zellfunktionen wie Proliferation, Zellzyklus und Migration beitragen können, sind die Src Tyrosin Kinasen (SFKs). Neben der Dephosphorylierung als aktivierendes Prinzip, ist auch eine Plasmamembranverankerung von entscheidender Bedeutung für die Aktivität der SFKs. Ein wichtiger Faktor hierbei spielt UNC119, das myristoylierte Proteine, unter die auch die SFKs fallen, in der Zelle transportiert. In dieser Arbeit wurde ein UNC119-Inhibitor entwickelt, mit dem der Einfluss der UNC119-Inhibition auf insbesondere Src gezeigt werden konnte. Desweiteren wurde dieser Inhibitor als dual-selektiv für UNC119 und PDEδ erkannt, wobei damit zum ersten Mal damit die Inhibition der Plasmamembranverankerung von myristoylierten und, im Falle von PDEδ, prenylierten Proteinen gleichzeitig untersucht werden konnte. Im zweiten Teil wurde der Hedgehog-Signalweg untersucht. Dieser ist unter anderem involviert in die Regeneration von Gewebe. Hierbei würde ein Coaktivator für das Studium von Regenerationsprozessen sinnvoll sein. Es wurde ein Dipeptid mit drei Stereogenen Zentren identifiziert. Dieser und andere auf der Struktur basierenden Diastereomere/Derivate stellten sich jedoch als biologisch instabil heraus, da wahrscheinlich die stereogenen Zentren in der Zelle isomerisieren können. Weiterhin wurde in dieser Dissertation eine γ-selektive, vinyloge C-Ferrier-Umlagerung optimiert. Hierbei wurden acetylierte Glucalderivate mit einem TBS-geschützten Dioxinon und Zinktriflat als Katalysator erfolgreich umgesetzt. Ebenso war eine Hochskalierung der Reaktion erfolgreich, womit sich diese Reaktion als robust auszeichnet.
Genetically encoded conformational probes for small GTPase activity
(2018) Brand, Simone; Goody, Roger S.; Bastiaens, Philippe
Small GTPases are guanosine nucleotide binding enzymes that exist in two states, being active in the guanosine-triphosphate- (GTP) and inactive in the guanosine-diphosphate- (GDP) bound state. Thus, these proteins act as binary molecular switches, allowing signaling exclusively in the GTP-bound state and function in a variety of cellular signaling processes, including cytoskeleton organization, membrane trafficking, nuclear transport, cell survival and proliferation. Due to their localization to different cellular compartments as well as their nucleotide- and localization-dependent activity, small GTPases are attractive targets for the development of biosensors. Recently, a new type of FRET-based sensors for small GTPase activity was developed. These conformational probes for small GTPase activity (COSGA) allow for the direct detection of conformational changes within the target protein and thereby determining the activity state of the small GTPase. The work presented in this thesis focused on the development of a second generation of COSGA probes to facilitate the preparation process and the application for cellular studies. To genetically encode the sensor design, stop codon suppression technique and intracellular fluorescence labeling were used. The first part of this work focused on the development of a genetically encoded Rab1b probe to establish the second generation sensor, whereas the aim of the second part was to apply the sensor design to Rheb. At the present time, no effector protein has been reported for Rheb, hindering the development of conventional probes to monitor Rheb activity spatiotemporally. Due to its role as an activator of mTORC1, a master regulator for cell metabolism, growth and proliferation, Rheb is a particularly interesting target. This work demonstrates the establishment of a genetically encoded Rheb sensor using the previously described conformational sensor design. This Rheb sensor allows visualization of spatiotemporal Rheb activity in living cells.
Spatial distributed phosphatome determines EGFR phosphorylation response
(2017) Mhamane, Amit; Bastiaens, Philippe; Musacchio, Andrea
Autocatalytic activation of Epidermal Growth Factor Receptor (EGFR) at the plasma membrane increases the sensitivity of the cell to extracellular growth factors but can also generate spontaneous receptor activation in the absence of stimulation. As a mechanism to control EGFR phosphorylation at the plasma membrane, receptor endocytosis and vesicular trafficking relocalizes activated, phosphorylated EGFR to perinuclear compartments rich in Protein Tyrosine Phosphatases (PTPs), such as PTPN1, which dephosphorylate and inactivate the receptor. Although the role of few PTPs in regulating EGFR phosphorylation is known, it is unclear how PTPs that are spatially segregated in distinct cellular compartments, modulate EGFR autocatalytic activation and hence its downstream signaling. Through quantitative imaging of EGFR phosphorylation upon genetic perturbations of classical PTPs and EGFR-PTP interactions, we identified endoplasmic reticulum (ER) associated PTPN2 and plasma membrane associated receptor-like PTPRG/J as strong, direct negative regulators of EGFR. Using single cell measurements of phosphorylation of the EGFR downstream signaling tyrosine residue Y1068, we generated a spatial-temporal reactivity map to identify local phosphatase activity. By negatively regulating EGFR phosphorylation, we deduced the role of PTPN2/PTPRJ in determining signal duration: a function that is coupled to vesicular trafficking. Furthermore, by maintaining the plasma membrane density of EGFR due to its interaction with ligandless EGFR and dephosphorylation of EGFR at Y1045 - a cCbl-ubiquitin ligase binding site, PTPN2 participates in a spatially established negative-feedback that is mediated by vesicular recycling. Through its activity on ligandless EGFR at plasma membrane, PTPRG regulates the autocatalytic activity of EGFR and influences the responsiveness of a cell to EGF dose. Altogether our findings indicate that by spatially segregating PTPs with different functional relationships to EGFR, the cell is able to sense and respond to its environment.
Dynamic regulation of autocatalytic EGFR activation
(2017) Baumdick, Martin; Bastiaens, Philippe; Goody, Roger S.
The dynamics of epidermal growth factor receptor (EGFR) signaling emerge from its recursive interactions with protein tyrosine phosphatases (PTPs) and autocatalytic receptor activation thereby determining cellular behavior including proliferation, migration and differentiation. The autocatalytic activation of EGFR causes an amplification of EGFR phosphorylation in response to extracellular signals, but its trade-off is spontaneous activation at high receptor densities even in the absence of ligand. Structural and molecular dynamics studies identified an allosteric activation mechanism upon EGF-induced receptor activation, but the molecular basis of autocatalytic EGFR activation remains unclear. To better understand autocatalysis we developed a Förster Resonance Energy Transfer (FRET)-based, conformational EGFR indicator (CONEGI) using genetic code expansion to monitor the conformational state of the tyrosine kinase domain (TKD) in living cells and relate it to EGFR activity. We show that EGFR monomers can adopt an active conformation that is stabilized upon Y845 phosphorylation. Since Y845 is an auto-phosphorylation site this creates a positive feedback loop generating an autocatalytic amplification mechanism. To counteract autonomous, autocatalytic EGFR activation in the absence of ligand, intrinsic and extrinsic safeguard mechanisms are required. Intrinsic auto-inhibitory structural features can be overcome by thermal conformational fluctuations allowing a sub-population of EGFR to adopt an active conformation. This autonomous, autocatalytic EGFR activity is counterbalanced by a spatial cycle that suppresses phosphorylation of Y845 on EGFR monomers by vesicular recycling through perinuclear areas with high PTP1B activity. EGF-binding induces receptor dimerization and phosphorylation of the c-Cbl docking Y1045 leading to receptor ubiquitination that targets EGFR for degradation in lysosomes. The re-routing regulates EGFR signaling response by the transit-time to late endosomes where it is switched-off by high PTP1B activity. This ubiquitin-mediated switch from a suppressive cycle to a unidirectional trafficking mode is a uniquely suited solution to suppress spontaneous activation while maintaining responsiveness to EGF.
Investigation of the properties of Acyl protein thioesterases and their role in Ras depalmitoylation
(2017) Baumeister, Stefan; Winter, Roland; Musacchio, Andrea
Es konnte gezeigt werden, dass die Inhibition des Proteins Acyl Protein Thioesterase (APT) die Lokalisierung von palmitoylierten Ras Proteinen derart verändern kann, dass eine Reduzierung der onkogenen Ras Aktivität beobachtet wird. Die genaue physiologische Rolle von APT ist allerdings weiterhin unklar, da sowohl weitere palmitoylierte Proteine, als auch (Lyso-) Phospholipide und Prostaglandin Glycerin Ester als Substrate von APT beschrieben wurden. Die hier vorliegende Arbeit liefert, durch Untersuchung der Interaktionen verschiedener Substrate, Liganden und Inhibitoren mit APT, neue Erkenntnisse bezüglich möglicher zellulärer Aufgaben von APT. Die gewonnenen Resultate helfen zu bestimmen, welche Substrate von APT bevorzugt werden, wie eine Substratspezifität erreicht wird und wie sich APT im Laufe der Evolution in verschiedenen eukaryotischen Spezies entwickelt hat.
Fluoreszenzbasierte Analyse von RNA-Protein-Interaktionen
(2017) Roszyk, Laura; Brakmann, Susanne; Summerer, Daniel
Evaluierung der Lipidmuster S-acylierter Proteine und Azido-Ceramid Derivate zur proteomweiten Identifizierung neuer C16- Ceramid bindender Proteine
(2017) Schulte-Zweckel, Janine; Waldmann, Herbert; Dehmelt, Leif
Part 01: A Bioorthogonal Probe for Quantitative Profiling of Protein S-Acylation We have developed a chemical probe that enables the accurate and systematic identification of Sbound fatty acids. The analysis of the S-acylome in different cell lines as well as in an enriched N-Ras revealed that proteins are modified with fatty acids of various chain lengths and structures. These previously overlooked fatty acid modifications may have a key role in regulating protein localization and function. Hence, atomic force microscopy studies and FRET-based kinetics assays indicated that the unsaturated N-Ras presents an increased tendency toward clustering and higher insertion kinetic rate constants compared to that of its saturated counterpart. The technique could be also applied to investigate the role of CLN3 as a potential fatty acid desaturase. Indeed, overexpression of CLN3 results in all the cases in a significant increase in C16:1 levels. This effect was most marked for N-Ras compared to the whole S-acylome, which it may be explained by the substrate specificity of CLN3 or its particular subcellular localization. We are convinced that the developed methodology may strongly contribute to a better understanding of S-acylated proteins. Part 02: Azido-tagged Sphingolipids for the proteome-wide identification of novel C16-ceramidebinding proteins To generate a candidate list of ceramide-interacting proteins we applied two methods, a mass spectrometry-based proteomic approach and a proteome microarray. 214 and 127 protein candidates were identified respectively, and 24 were found in both data sets. We initially chose potential candidates for testing on the basis of its potential implication in ceramide-mediated pathways. Binding could be confirmed for: AIFm2, APP, BUD31, Gal-1 and Gal-3, mTOR and PPT1. The specificity of those proteins for ceramide species with different chain lengths has been also analysed using a pull-down and a competition assay, observing a dose-dependent decrease in the interaction of the lipid with the studied proteins. To further validate the binding of ceramide to the identified proteins we employed a cellular thermal shift assay. Treatment of cell lysates with C16-Cer resulted in a slighter stabilization of APP, BUD31 and PPT1 and an appreciable increase of melting point temperatures for the other investigated proteins. Finally, the direct interaction of C16-Cer was investigated in vitro using a DSF and recombinantly expressed proteins. AIFm2, BcL-xL, Gal-1 were selected and direct interaction with variable affinities could be proved in all the cases, whereas no binding could be observed when AIFm2 and Gal-1 were titrated with increasing concentrations of palmitic acid, or when C-16-Cer was tested against Son of Sevenless (SOS) protein as a negative control. Future experiments will be required to elucidate how ceramide binding affects protein activity, if it relies on the modulation of protein localization such as the targeting of LC3B to mitochondria, or if, in turn, it directly regulates protein function such as the activation of the protein phosphatase PP2A.
Mechanistic studies of Rab GTPase membrane targeting and cycling in cells
(2017) Li, Fu; Goody, Roger S.; Bastiaens, Philippe
Small molecule inhibition of lipidated proteins/cargo interaction and synthesis of a cinchona alkaloid-derived library as potent autophagy inhibitors
(2017) Garivet, Guillaume; Waldmann, Herbert; Wittinghofer, Alfred
The effect of oncogenic HRas on the RTK-PTP reaction network
(2017) Stockert, Rabea; Bastiaens, Philippe; Goody, Roger S.
Discovery and target identification of small molecule autophagy inhibitors
(2017) Robke, Lucas; Waldmann, Herbert; Engelhard, Martin
Pharmacologically active substances have been applied in medicine for at least 5300 years.Ever since, mankind has tried to enlarge the assortment of bio-active substances, in order to fight diseases and ultimately maintain health and quality of life. For thousands of years, but especially in the last century, drug discovery has been the scientific key driver for the progression of medicine. One process that attracts notable attention due to its high potential for the development of new drugs is autophagy. Autophagy is a catabolic process which plays a crucial role in the development, differentiation, homeostasis and survival of cells. It mediates the degradation of cellular components especially under conditions such as starvation, and the misregulation of autophagy is implicated in various diseases, ranging from cancer to microbial infections and neurodegenerative diseases. Small organic molecules still account for 90% of today’s pharmaceuticals and drug-like modulators of biological processes have proven to be valuable reagents and tools for chemical biology. However, to date only a few modulators of autophagy have been reported. Therefore the goal of this work was to identify and characterize new autophagy inhibitors. A cell-based assay was performed to identify inhibitors of autophagic flux. Based on hits from the cellular assay compound libraries were synthesized to provide information regarding structure-activity relationships. The identification of the targets of the inhibitors was achieved through affinity chromatography, mechanistic reasoning and the process of elimination. The examination of the targets’ roles in autophagy enlarged our understanding of the autophagic process and allowed us to evaluate their potential as new drug targets. Ultimately, the identified and characterized novel chemotypes for autophagy inhibition provide valuable tools to study their target protein’s function as well as the autophagic process more precisely. They can furthermore provide promising starting points for drug discovery focusing on cancer.
Knock off and on: a novel approach to autophagy modulation
(2017) Klewer, Laura Kristin; Goody, Roger S.; Bastiaens, Philippe
Identification and biological characterization of indoline-based autophagy inhibitors
(2017) Rummelt, Marjorie A.; Waldmann, Herbert; Engelhard, Martin
Autophagy is a vital catabolic process involved in the degradation of cellular components, which plays an important role in cancer and neurodegenerative disorders. Due to their high complexity, the pathways regulating autophagy have not been fully elucidated. The development of specific autophagy modulators and the identification of their cellular targets can provide valuable insight into the regulation of this process and, ultimately, its role in disease. Herein, a potent inhibitor of starvation-induced autophagy (IC50 = 520 +- 550 nM) was identified using a phenotypic screen. In a series of secondary assays, the compound was validated as an autophagy inhibitor. The structure–activity relationship for this compound class was derived from a synthesized compound collection of structural analogs and used for the preparation of a chemical probe for affinity-based proteomics ("pull-down"). Glutamate dehydrogenase (GDH) and calpain-1 were isolated by means of affinity-based proteomics but devalidated as targets of the inhibitor in various biophysical and biochemical assays. The ATP-gated ionotropic receptor P2X4 and the Ragulator component LAMTOR5 were the most promising targets determined by thermal proteome profiling and are currently under evaluation.Targets predicted using a computational approach based on chemical similarity included 5-hydroxytryptamine (5-HT) receptors and multiple kinases—rapidly accelerated fibrosarcoma (RAF1) and related kinases, pyruvate kinase (PK), and abelson murine leukemia viral oncogene homolog 1 (ABL1). Whereas no modulating effect on the predicted kinases was observed, the autophagy inhibitor was a potent antagonist of the G-protein-coupled serotonin receptor 5-HT6 (IC50 = 1 µM). However, as known antagonists of the receptor did not inhibit autophagy, 5-HT6 was considered an off-target. Given the strong antagonistic effect seen, G-protein-coupled receptors need to be further evaluated as potential targets of the identified inhibitor. The successful target identification of this potent inhibitor could help deliver further understanding of the complex mechanisms that regulate autophagy.
Identifizierung und Charakterisierung neurotropher Naturstoffderivate und kovalenter Kinesininhibitoren
(2017) Förster, Tim; Waldmann, Herbert; Dehmelt, Leif
Die Neugestaltung des neuronalen Netzwerkes ist essentiell für die Wiederherstellung der physiologischen Funktionen nach neuronaler Schädigung, weshalb die Erforschung neuartiger Therapeutika mit neurotrophen Eigenschaften von großer Bedeutung ist. In dieser Arbeit wurde ein Testsystem zur Identifizierung neuromodulatorischer Substanzen in der Neuroblastomzelllinie SH-SY5Y entwickelt. Durch Anwendung dieses Testsystems konnten neurotrophe Verbindungen auf Basis der Naturstoffe Militarinon A und (Iso-) Rhynchophyllin identifiziert werden. Da über die Wirkmechanismen neuroaktiver Pyridonalkaloide bisher wenig bekannt ist, geben die Ergebnisse neue Einblicke in die modulierten Prozesse von biologisch aktiven Vertretern dieser Substanzklasse. Ebenso konnten durch die neurotrophen Indolalkaloide weitere Erkenntnisse in Hinblick auf die neuropharmakologischen Eigenschaften dieser Moleküle gewonnen werden. Das Kinesin HSET gilt als vielversprechendes Zielprotein bei der Erforschung neuartiger Wirkstoffe zur Behandlung von Krebserkrankungen. HSET besitzt eine essentielle Funktion bei der zentrosomalen Gruppierung in Tumorzellen mit amplifizierten Zentrosomen, während in gesunden Zellen funktionell dominante Zentrosomen vorhanden sind. Daher besitzen Inhibitoren dieses Kinesins das Potential, spezifisch Tumorzellen zu attackieren. In dieser Arbeit wurde ein Sulfonylpyrimidin als erster kovalenter HSET-Inhibitor identifiziert, der weitere Möglichkeiten zur Entwicklung von Therapeutika gegen Tumorzellen mit amplifizierten Zentrosomen eröffnet.
Zellgängige Peptide zur Inhibition des Wnt/β-Catenin Signalweges
(2017) Dietrich, Laura; Brakmann, Susanne; Grossmann, Tom N.
Der Wnt/β-Catenin Signalweg kontrolliert fundamentale Prozesse der Embryonalentwicklung sowie die Homöostase im adulten Gewebe. Fehlregulation durch konstitutive Aktivierung resultiert in übermäßiger Proliferation und fördert die Entstehung von Krebs. Die Inhibition des Signalweges ist sehr anspruchsvoll, da dieser durch Protein-Protein-Interaktionen (PPIs) reguliert wird, die nur schwer mit niedermolekularen Verbindungen moduliert werden können. Peptide weisen gute Bindungseigenschaften für flache und ausgedehnte Interaktionsflächen auf und bilden die Grundlage neuartiger Strategien zur Adressierung von PPIs. In dieser Arbeit wurde eine Strategie zur Optimierung der Bioverfügbarkeit eines stapled peptides zur Inhibition der β-Catenin/TCF-Interaktion entwickelt. Aus der vergleichenden Analyse mit bekannten zellgängigen Peptiden und stapled peptides wurde durch feine Nuancierung von Hydrophobizität, Nettoladung und Ladungsverteilung NLS StAx h erhalten. NLS StAx h inhibiert sowohl selektiv die Interaktion zwischen β Catenin und TCF, als auch spezifisch die Proliferation und Migration von Darmkrebszellen. In einem ex vivo Darmkrebsmodell wurde außerdem nachgewiesen, dass die mit Darmkrebs assoziierte abnormale Genexpression auf das Basallevel reduziert wurde. NLS-StAx-h ist damit die erste Verbindung, die eine gute zelluläre Aufnahme mit einer effizienten Hemmung der β-Catenin/TCF-Wechselwirkung kombiniert.
Identifizierung neuer PDE6D-Bindungspartner und Entwicklung eines phänotypischen Screens zur Identifizierung biologisch aktiver Indolochinolizine
(2017) Küchler, Philipp Robert Felix; Waldmann, Herbert; Dehmelt, Leif
Die Protein-Prenylierung ist eine posttranslationale Modifikation, die die Affinität von Proteinen gegenüber biologischen Membranen erhöht und Protein-Protein-Interaktionen (PPI) vermitteln kann. Das molekulare Chaperon PDE6D (Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit delta) ist sowohl für die korrekte Membranlokalisation prenylierter kleiner GTPasen als auch für deren Solubilisierung im wässrigen Milieu des Zytosols verantwortlich. Für das farnesylierte Protoonkogen KRAS konnte ein PDE6D-abhängiger Transport an die Plasmamembran gezeigt werden. Die spezifische Inhibition dieser Interaktion mittels niedermolekularer Verbindungen führt zur Relokalisation von KRAS an das Endomembransystem, einhergehend mit einer verringerten Aktivierung des MAPK/ERK-Signalwegs und einer Wachstumsinhibition von KRAS-abhängigen Pankreastumorzellen. Aufgrund des Missverhältnisses zwischen der Größe des humanen Prenyloms (ca. 100 – 500 Proteine) und der Anzahl bekannter PDE6D-Bindungspartner wurde innerhalb dieser Arbeit eine affinitätschromatographische Strategie mit anschließender massenspektrometrischer Analyse entwickelt, mit deren Hilfe neue, bisher unbekannte prenylabhängige Bindungspartner PDE6Ds identifiziert werden konnten. Als neue Interaktoren konnten die RHO-GTPase CDC42, die kleine GTPase RAB23 sowie die Phosphodiesterase CNP nachgewiesen werden. Die Wechselwirkung mit allen drei Proteinen konnte durch Verwendung der PDE6D-Inhibitoren Deltarasin und Deltazinone 1 gestört und die Auswirkungen auf die subzelluläre Lokalisation der Proteine in Zellen untersucht werden. Weiterhin konnte als neuer Interaktor PDE6Ds die permanent farnesylierte Lamin A-Mutante Progerin identifiziert werden. Die seltene genetische Störung Hutchinson-Gilford Progeria Syndrom (HGPS) wird in ca. 90% der Fälle durch eine heterozygote de novo Mutation hervorgerufen, die in der Folge zur Expression des dominant negativen Genprodukts Progerin führt. Ursächlich für den durch die Progerinexpression hervorgerufenen Phänotyp ist die im Unterschied zum Wildtyp-Protein permanente Farnesylierung, die Auswirkungen auf die Morphologie und Integrität des Zellkerns hat. Durch die Untersuchung von Prenylierungsmutanten konnte die prenylabhängige Interaktion zwischen Progerin und PDE6D sowohl in Zelllysat als auch – mittels PLA-Technologie (proximity ligation assay) – in Zellen nachgewiesen werden. Weiterhin konnte die Progerin-PDE6D-Interaktion durch Deltarasin und Deltazinone 1 inhibiert werden. Neben den Auswirkungen einer Störung dieser Interaktion mittels niedermolekularer Verbindungen wird in der vorliegenden Arbeit auch die Bindungsspezifität PDE6Ds gegenüber prenylierten Cargoproteinen unter Berücksichtigung der hier erhaltenen Ergebnisse diskutiert. Der zweite Teil der Arbeit beschäftigt sich mit der Identifikation biologisch aktiver Naturstoffderivate, die humane Zellen während der Zellteilung arretieren. Die Akkumulation von Mutationen in Krebszellen führt zu unplanmäßigen Teilungsvorgängen und genomischer Instabilität. Diverse therapeutische Strategien zur Adressierung des Zellzyklus in Krebszellen wurden bereits untersucht. Viele dieser Strategien konnten jedoch nicht den gewünschten therapeutischen Erfolg einhergehend mit geringen zytotoxischen Effekten auf gesunde Zellen erreichen. Die Wirkungsweise der meisten Zellzyklus-adressierenden Substanzen beruht dabei auf der Aktivierung des Spindelkontrollpunkts (SAC, spindle assembly checkpoint). Die chronische Aktivierung des Kontrollpunkts und die damit einhergehende Arretierung der Zelle in der Metaphase durch chemische Agenzien bestimmt dabei zwei mögliche Schicksale für die Zelle; Entweder die Zelle teilt sich trotz aktiviertem Kontrollpunkt in Tochterzellen in einem Vorgang, der als „mitotic slippage“ bezeichnet wird und eventuelle Aneuploidien zur Folge haben kann, oder die Zelle stirbt einen apoptotischen Zelltod während der Mitose. Innerhalb der Abteilung „Chemische Biologie“ (MPI Dortmund) wurde mithilfe eines vorwärts gerichteten chemisch-genetischen Ansatzes das biologisch aktive Indolochinolizin Centrocountin-1 entwickelt, das über Interaktion mit den Zielproteinen Nucleophosmin (NPM) und Exportin-1 (CRM1) zu einer Aktivierung des Spindelkontrollpunkts und einem vermehrten apoptotischen Zelltod führt. In dieser Arbeit konnte ein zellbasierter phänotypischer Screen entwickelt werden, der zur Identifizierung neuer aktiver Centrocountin—1-Derivate verwendet wurde. Mittels automatischer Mikroskopie und anschließender Quantifizierung der gewonnenen Bilddaten konnte aus einer kleinen Bibliothek an Centrocountin-Derivaten das Pyridoisochinolizin a528 als potenteste Substanz identifiziert werden. Folgeversuche zeigten eine zweifache Erhöhung des mitotischen Index im Vergleich zu Centrocountin-1 und stärkere Auswirkungen auf die Proliferation von HeLa-Zellen als die Ursprungssubstanz. Des Weiteren wurde im Rahmen dieser Arbeit der Wirkmechanismus der Centrocountine weiter erörtert. Aufgrund der diversen Funktionen von NPM hinsichtlich Chaperonaktivität, DNA-Reparatur, Ribosomenbiogenese und Zentrosomen-Integrität ergeben sich verschiedene Möglichkeiten eines Einflusses von Centrocountin-1. In diesem Zusammenhang konnte neben einem Einfluss von Centrocountin-1 auf die Oligomerisierung von NPM auch die aus RNAi-Studien bekannte Häufung von DNA-Schäden in NPM-defizienten Zellen ausgeschlossen werden. Störungen des CRM1-abhängigen Transports von FOXO3 bestätigen vorherige Ergebnisse zu einem Einfluss von Centrocountin-1 auf den Kernexportfaktor CRM1. In dieser Arbeit werden die gewonnenen Ergebnisse in Hinblick auf den potentiellen Wirkmechanismus der Centrocountine diskutiert.
Systems analysis of the spatial regulation of oncogenic Ras signalling
(2017) Heinelt, Kaatje Friederike; Bastiaens, Philippe; Dehmelt, Leif
Ras (Rat sarcoma) isoforms are small GTP-binding proteins that play a major role in the signalling networks controlling cell growth and survival. The Kras isoform is of particular interest as in many severe kinds of cancer the presence of oncogenic Kras mutations is associated with a poor prognosis. Kras is associated with the plasma membrane due to its farnesyl moiety and a polybasic motif and functions as a signalling hub. If Kras gets lost from the plasma membrane due to spontaneous dissociation or endocytosis, it will equilibrate over the extensive endomembrane system inside the cell. With Kras no longer present at the plasma membrane, its activation and the following activation of subsequent pathways can no longer take place. However, to remain on the plasma membrane, Kras has to be constantly enriched there. This enrichment must be actively maintained in the cell by an energy-driven mechanism involving the solubilising factor PDEδ. Consequently, inhibition or down-modulation of PDEδ results in mislocalisation of Kras, making PDEδ an interesting target for anti-cancer drug development. In 2013 a small molecule, Deltarasin, was identified as a potent inhibitor of PDEδ causing a redistribution of Kras from the plasma membrane towards endomembranes when applied to cells. This work investigates whether small molecule PDEδ inhibitors such as Deltarasin affect (K) Ras localization in space and time. It demonstrates that PDEδ inhibition causes Ras relocalisation from the plasma membrane towards the endomembranes in different human cancer cell lines and in murine small intestine organoids, which express endogenous levels of oncogenic Kras. Nonetheless, it has been shown that Deltarasin has certain side effects, e.g. it becomes cytotoxic at higher concentrations. Hence, a new PDEδ inhibitor, Deltazinone 1, which is supposed to be less cytotoxic in comparison to Deltarasin, was synthesized. In order to determine whether it represents a viable alternative to Deltarasin, its ability to relocalise Kras in a panel of cancer cell lines was tested and it was indeed possible to mislocalise Kras with this inhibitor in Kras-dependent PancTuI cells. Deltazinone 1 and Deltarasin both had a demonstrable effect on cell growth/survival, respectively. In this way it appears that PDEδ constitutes a valid target for the pharmacological therapy of Kras-dependent tumours. This work demonstrates that two specific PDEδ inhibitors with completely different lead structures are capable of mislocalising Kras to endomembranes. In sum, it demonstrates that the availability of PDEδ is essential to ensure (K) Ras localization at the plasma membrane in Kras-dependent cancer cells and thus that the survival of those cells are ultimately dependent on PDEδ.

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