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Transfer RNAs (tRNAs) are highly structured non-coding RNAs which play key roles in translation and cellular homeostasis. tRNAs are initially transcribed as precursor molecules and mature by tightly controlled, multistep processes that involve the removal of flanking and intervening sequences, over 100 base modifications, addition of non-templated nucleotides and aminoacylation. These molecular events are intertwined with the nucleocy- toplasmic shuttling of tRNAs to make them available at translating ribosomes. Defects in tRNA processing are linked to the development of neurodegenerative disorders. Here, we summarize structural aspects of tRNA processing steps with a special emphasis on intron-containing tRNA splicing involving tRNA splicing endonuclease and ligase. Their role in neurological pathologies will be discussed. Identification of novel RNA substrates of the tRNA splicing machinery has uncovered functions unrelated to tRNA processing. Future structural and biochemical studies will unravel their mechanistic underpinnings and deepen our understanding of neurological diseases.
The KMT2A (MLL) gene rearrangements (KMT2A-r) are associated with a diverse spectrum of acute leukemias. Although most KMT2A-r are restricted to nine partner genes, we have recently revealed that KMT2A-USP2 fusions are often missed during FISH screening of these genetic alterations. Therefore, complementary methods are important for appropriate detection of any KMT2A-r. Here we use a machine learning model to unravel the most appropriate markers for prediction of KMT2A-r in various types of acute leukemia. A Random Forest and LightGBM classifier was trained to predict KMT2A-r in patients with acute leukemia. Our results revealed a set of 20 genes capable of accurately estimating KMT2A-r. The SKIDA1 (AUC: 0.839; CI: 0.799–0.879) and LAMP5 (AUC: 0.746; CI: 0.685–0.806) overexpression were the better markers associated with KMT2A-r compared to CSPG4 (also named NG2; AUC: 0.722; CI: 0.659–0.784), regardless of the type of acute leukemia. Of importance, high expression levels of LAMP5 estimated the occurrence of all KMT2A-USP2 fusions. Also, we performed drug sensitivity analysis using IC50 data from 345 drugs available in the GDSC database to identify which ones could be used to treat KMT2A-r leukemia. We observed that KMT2A-r cell lines were more sensitive to 5-Fluorouracil (5FU), Gemcitabine (both antimetabolite chemotherapy drugs), WHI-P97 (JAK-3 inhibitor), Foretinib (MET/VEGFR inhibitor), SNX-2112 (Hsp90 inhibitor), AZD6482 (PI3Kβ inhibitor), KU-60019 (ATM kinase inhibitor), and Pevonedistat (NEDD8-activating enzyme (NAE) inhibitor). Moreover, IC50 data from analyses of ex-vivo drug sensitivity to small-molecule inhibitors reveals that Foretinib is a promising drug option for AML patients carrying FLT3 activating mutations. Thus, we provide novel and accurate options for the diagnostic screening and therapy of KMT2A-r leukemia, regardless of leukemia subtype.
Die Fähigkeit der spezifischen und kontextabhängigen zellulären Adaption auf intrinsische und/oder extrinsische Signale ist das Fundament zellulärer Homöostase. Verschiedene Signale werden von Membranrezeptoren oder intrazellulären Rezeptoren erkannt und ermöglichen die molekulare Anpassung zellulärer Prozesse. Komplexe, ineinandergreifende Proteinnetzwerke sind dabei elementar in der Regulation der Zelle. Proteine und deren Funktionen werden dabei nach Bedarf reguliert und unterliegen einem ständigen proteolytischen Umsatz.
Die stimulusabhängige Gentranskription und/oder Proteintranslation nimmt hier eine zentrale Stellung ein, da die zugrundeliegende Maschinerie die Komposition und Funktion der Proteinnetzwerke entsprechend anpassen kann. Zusätzlich zur Regulation der Proteinabundanz werden Proteine posttranslational modifiziert, um deren Eigenschaften rasch zu ändern. Zu posttranslationalen Modifikationen zählen die Ubiquitinierung und/oder Phosphorylierung, welche die Proteinfunktionen hochdynamisch regulieren. Deregulierte Proteinnetzwerke werden oft mit Neurodegeneration und Autoimmun- oder Krebserkrankungen assoziiert. Auch Infektionen mit humanpathogenen Bakterien greifen stark in den Regulierungsprozess von Proteinnetzwerken und deren Funktionen ein. Die zelluläre Homöostase wird dadurch herausgefordert.
Bakterien der Gattung Salmonella sind zoonotische, gramnegative, fakultativ intrazelluläre Pathogene, welche weltweit millionenfach Salmonellen-erkrankungen hervorrufen. Von besonderer Bedeutung ist dabei Salmonella enterica serovar Typhimurium (hiernach Salmonella), welches im Menschen, meist durch mangelnde Hygienemaßnahmen, Gastroenteritis auslöst.
Immunität in Epithelzellen wird über das angeborene Immunsystem vermittelt und dient der Pathogenerkennung und -bekämpfung. Die Toll-like Rezeptoren (TLR) gehören zu den Mustererkennungsrezeptoren (pattern recognition receptors), welche spezifische mikrobielle Strukturen detektieren und eine kontextabhängige zelluläre Antwort generieren. Danger-Rezeptoren erkennen hingegen nicht direkt das Pathogen, sondern zelluläre Perturbationen, welche durch Zellschäden oder bakterielle Invasionen verursacht werden. Die intrinsische Fähigkeit der Wirtszelle, sich gegen Infektionen/Gefahren zu wehren wird dabei als zellautonome Immunität bezeichnet. Dabei nehmen induzierte proinflammatorische Signalwege und zelluläre Stressantworten eine wichtige Stellung ein. Die zelluläre Stressantwort aktiviert unter anderem die selektive Autophagie. Diese kann spezifisch aberrante Organelle, Proteine und invasive Pathogene abbauen. Ein weiterer Stresssignalweg ist die integrated stress response (ISR), welche eine selektive Proteintranslation erlaubt und damit die Auflösung des proteintoxischen Stresses ermöglicht.
Zur Penetration von Epithelzellen benötigt Salmonella ein komplexes System an Virulenzfaktoren, welches die bakterielle Internalisierung und Proliferation in der Wirtszelle ermöglicht. Salmonella nutzt dazu ein Typ-III-Sekretionssystem. Das System sekretiert bakterielle Virulenzfaktoren in die Zelle, sodass eine hochspezifische Modulierung des Wirtes erzwungen wird.
Die Virulenzfaktoren SopE und SopE2 spielen dabei eine Schlüsselrolle, da sie die Pathogenität von Salmonella maßgeblich vermitteln. Durch molekulare Mimikry von Wirts GTP (Guanosintriphosphat) -Austauschfaktoren aktivieren SopE und SopE2 die Rho GTPasen CDC42 und Rac1. GTP-geladenes CDC42 und Rac1 wiederum aktivieren das Aktinzytoskelett und stimulieren die Polymerisierung von Aktinfilamenten über den Arp2/3-Komplex an der Invasionsstelle. Das Pathogen wird dadurch in ein membranumhülltes Vesikel, die sogenannte Salmonella-containing Vakuole (SCV), aufgenommen. Die SCV stellt eine protektive, replikative, intrazelluläre Nische des Pathogens dar und wird permanent durch verschiedene Virulenzfaktoren moduliert.
Im Allgemeinen führt die Aktivierung von Mustererkennungsrezeptoren und Danger-Rezeptoren also zu einer zellulären Stressantwort und Entzündungsreaktion, wodurch es zur Bekämpfung der Infektion kommt. Inflammatorische Signalwege werden meist über den zentralen Transkriptionsfaktor NF-κB (nuclear factor 'kappa-light-chain-enhancer' of activated B-cells) vermittelt. NF-κB bewirkt die Induktion von proinflammatorischen Effektoren und Stressgenen. Zellautonome Immunität wird zusätzlich durch antibakterielle Autophagie ermöglicht, wobei Salmonella selektiv über das lysosomale System abgebaut werden. Das bakterielle Typ-III-Sekretionssystem verursacht an einigen wenigen SCVs Membranschäden, sodass Salmonella das Wirtszytosol penetrieren. Zytosolische Bakterien werden dabei spezifisch ubiquitiniert. Dies erlaubt die Erkennung durch die Autophagie-Maschinerie.
In der vorliegenden Arbeit wurde die zellautonome Immunität von Epithelzellen während einer akuten Salmonella Infektion durch quantitative Proteomik untersucht...
Die vorliegende Dissertation stellt eine Methode zur Löslichkeitsbestimmung vor, die für die Anwendung im Rahmen von BCS-Biowaiver Monografien entwickelt wurde. Der Methode und dem dafür konzipierten Studienprotokoll liegt das Prinzip der „Minimallöslichkeit“ zugrunde. Damit lässt sich einfach, kosteneffizient und wissenschaftlich verlässlich feststellen, ob ein Arzneistoff „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien der Gesundheitsbehörden FDA, EMA und WHO ist und sich dementsprechend generische Produkte des Arzneistoffs grundsätzlich für das BCS-Biowaiver Zulassungsverfahren eignen.
Dieses Verfahren für die Zulassung von Generika erlaubt die Beurteilung der Bioäquivalenz eines festen generischen Arzneimittels zur peroralen Anwendung auf Basis von in vitro-Freisetzungsuntersuchungen anstatt von in vivo-Studien wie z.B. pharmakokinetischen Studien am Menschen und erleichtert dadurch eine Marktzulassung sowohl durch Zeit- als auch Kosteneinsparung. Die Anwendung des Verfahrens ist von Vorteil, um die Verfügbarkeit von qualitativ hochwertigen, generischen (und damit kostengünstigen) Arzneimitteln zu erhöhen. Dies ist besonders wünschenswert für die Verfügbarkeit von gemäß der Weltgesundheitsorganisation essenziellen Arzneistoffen und unter denen gerade von solchen, die zur Bekämpfung von Krankheiten mit nur wenigen und/oder teuren therapeutischen Alternativen benötigt werden.
Entstanden ist die Löslichkeitsbestimmungsmethode im Rahmen von zwei Projekten, die beide zu diesem Ziel einer guten globalen Gesundheitsversorgung beitragen: die Erstellung der Biowaiver Monografien von Proguanilhydrochlorid (ein Malaria-Prophylaktikum) und Cefalexinmonohydrat (ein Antibiotikum aus der Gruppe der Cephalosporine) setzt die Publikationsreihe „Biowaiver Monograph Series“ der FIP Focus Group „Bioclassification/Biowaiver“ fort. Jede Monografie gibt eine umfassende wissenschaftliche Empfehlung zur Eignung eines Wirkstoffs der WHO „Model List of Essential Medicines“ und seiner generischen Produkte für das BCS-Biowaiver Verfahren hinsichtlich aller regulatorisch geforderten Aspekte ab. Proguanilhydrochlorid (BCS Klasse III – „hochlöslich“ und nicht „hoch permeabel“) und Cefalexinmonohydrat (BCS Klasse I – „hochlöslich“ und „hoch permeabel“) sind beide für dieses Zulassungsverfahren geeignet.
Im Zuge des anderen Projektes wurde die Löslichkeit und anschließend die BCS Klasse von Wirkstoffen bestimmt, die der 16. und 17. Version der WHO „Model List of Essential Medicines“ neu hinzugefügt wurden. Neun von 16 untersuchten Wirkstoffen, die in feste, perorale Arzneimittel formuliert werden können, sind im Hinblick auf ihre BCS Klasse für das eine Zulassung per BCS-Biowaiver geeignet. Eine umfangreichere Empfehlung könnte im Rahmen einer Biowaiver Monografie gegeben werden.
Die experimentelle Bestimmung der Löslichkeit über einen pH-Wert-Bereich von 1-6,8 war essenzieller Bestandteil beider Projekte, da Literaturdaten zur Löslichkeit der Wirkstoffe nicht oder nur unvollständig vorlagen. Die entwickelte Methode basiert auf einer im Kleinmaßstab angesetzten „Shake-Flask“-Methode zur Bestimmung der thermodynamischen Löslichkeit, wird jedoch in einem Zeitrahmen von 24 Stunden durchgeführt. Sie nutzt die höchste Dosis der Wirkstoffe als Substanzmenge, um zu bestimmen, ob dieser „hochlöslich“ gemäß den BCS-Biowaiver Richtlinien ist oder nicht. Die Methode bzw. das dazugehörige Studienprotokoll beinhalten Empfehlungen zu den einzelnen Schritten der Durchführung, der Auswahl der Medien und Herausforderungen wie Präzipitation (Fallbeispiel: Proguanilhydrochlorid) und Zersetzungsreaktionen (Fallbeispiel: Cefalexinmonohydrat). Löslichkeitsdaten, die mit dieser Methode erhoben werden, können für eine Zulassung per BCS-Biowaiver bei den Gesundheitsbehörden eingereicht werden, aber auch für ein Vorab-Screening genutzt werden, dass „hochlösliche“ Arzneistoffe aus einer Vielzahl von Substanzen herauszufiltern soll, um nähere Untersuchungen im Rahmen einer Biowaiver Monografie anzuschließen.
Im Rahmen dieser vorliegenden Thesis wurden verschiedene photosensitive Systeme anhand statischer und zeitaufgelöster optischer Spektroskopiemethoden charakterisiert. Das Hauptaugenmerk dieser Arbeit lag in der Entwicklung und Untersuchung neuer Quantenpunkt-basierter Hybridsysteme. Es war möglich die optischen Eigenschaften der Quantenpunkte über Optimierung der Syntheseschritte zu variieren und so auf geplante Projekte anzupassen.
Im Projekt „Quantenpunkte als Zwei-Photonen Antenne“ sollten die hohen Zwei-Photonen Einfangquerschnitte von Quantenpunkten ausgenutzt werden um in Kombination mit einer photolabilen Schutzgruppe, ein Uncaging im NIR-Bereich zu realisieren. Es wurden ZnSe/ZnS Partikel synthetisiert, die eine starke Emission im Bereich der Absorption der Schutzgruppe zeigen. Anhand von zeitaufgelösten transienten Absorptionsexperimenten mit einer Anregungswellenlänge bei 775 nm wurde eine Zwei-Photonen Absorption der Partikel nachgewiesen. Jedoch wurden starke Emissionsbeiträge aus Fallenzuständen und eine geringe Stabilität beobachtet. Die Synthese von CdS/ZnS Quantenpunkten lieferte stabile Partikel mit geringer trap state Emission. Diese Partikel wurden in einem Modellhybridsystem als Energiedonoren eingesetzt. Als Akzeptor wurde der Farbstoff Cumarin343 gewählt. In statischen Absorptions- und Emissionsmessungen, zeitkorrelierten Einzelphotonenmessungen sowie in fs-zeitaufgelösten transiente Absorptionsmessungen konnte ein ultraschneller Energietransfer nach Ein-Photonen Anregung des Hybridsystems beobachtet werden. Über TPiF Messungen wurde die Zwei-Photonen Absorption der Quantenpunkte detektiert. Ein Energietransfer nach Zwei-Photonen Anregung der Quantenpunkte wurde beobachtet. Schließlich wurde ein Hybridsystem aus CdS/ZnS und der photolabilen Schutzgruppe Az-NDBF (Synthese im AK Heckel, Goethe Universität, Frankfurt a. M.) untersucht. Auch in diesem System wurde ein Energietransfer von Quantenpunkt auf die Schutzgruppe nach Ein- und Zwei-Photonen Anregung beobachtet. Anhand von TA Experimenten wurde eine Zeitkonstante von <100 ps für den Energietransfer nach Ein-Photonen Anregung ermittelt. Es konnte anhand der vorgestellten Resultate gezeigt werden, dass sich Quantenpunkte, aufgrund der guten Anpassung ihrer optischen Eigenschaften generell sehr gut als Antennen für organische Verbindungen eigenen.
Des Weiteren wurde ein Hybridsystem aus CdSe/ZnS Quantenpunkten und einer Dyade (Verbindung eines DTE Photoschalters und BODIPY Derivats), entworfen und charakterisiert. Ein ultraschneller EET von BODIPY auf den geschlossenen DTE Schalter wurde in vorangegangenen Studien beobachtet. Dieser EET führte zur Löschung der BODIPY-Emission. Sobald der Photoschalter im offenen Zustand vorliegt, findet aufgrund des fehlenden spektralen Überlapps kein EET statt und es wird die BODIPY-Emission detektiert. Die Erweiterung der Dyade um einen Quantenpunkt zeigte nach Anregung des Quantenpunkts dessen Fluoreszenzlöschung. Da die Emissionsbande der Quantenpunkte im Absorptionsbereich des BODIPY Farbstoffes liegt, konnte über statische und zeitaufgelöste Experimente ein ultraschneller EET von CdS/ZnS auf den Farbstoff ermittelt werden. Dies führte zu der Erweiterung des Anregungsspektrums des BODIPY Farbstoffs. Die Kopplung der Dyade an die Quantenpunktoberfläche lieferte eine Verbindung mit dem breiten Anregungsspekrum des Quantenpunkts und der schaltbaren Fluoreszenz der Dyade.
Das Hybridsystem aus CdSe Quantenpunkten und PDI zeigte vom Verhältnis der Quantenpunkte zu gekoppelten PDI Molekülen abhängige Fluoreszenzsignale. In TA Experimenten wurde ein ultraschneller EET ermittelt. Für hohe PDI Konzentrationen wurde ein weiterer EET von höher angeregten Elektronen auf das PDI identifiziert. Neben der EET Charakterisierung konnte ein zusätzlicher Prozess innerhalb des Hybridsystems mit hoher PDI Konzentration beobachtet werden. Auf den EET von Quantenpunkt auf PDI folgt ein ET aus dem Valenzband des Quantenpunkts in das HOMO des PDI*. In vorangegangene Arbeiten zu Hybridsystemen aus CdSe/ZnS und PDI wurde kein ET beobachtet. In dem beschriebenen Projekt konnte der Einfluss einer passivierenden Schale auf die elektronischen Eigenschaften von CdSe Quantenpunkten gezeigt werden.
Im letzten Teil dieser Thesis wurde die spektroskopische Charakterisierung einer NVOC und zweier NDBF Schutzgruppen beschrieben. Es konnten anhand statischer Absorptionsmessungen eine Freisetzungsquantenausbeute für NVOC-Adenin von 1,1 % ermittelt werden. Die Charakterisierung der Schutzgruppen mit einer NDBF Grundstruktur (DMA-NDBF und Az-NDBF) ergab eine Abhängigkeit der Freisetzungs- und Fluoreszenzausbeute von der Polarität des Lösungsmittels. In polarer Umgebung reduzierten sich die Quantenausbeuten deutlich...
The most versatile tool for visualizing endogenous RNA is molecular beacons (MBs). MBs are modified oligonucleotides that consist of a stem-loop structure equipped with a fluorophore and a quencher at the opposite ends. They only give a fluorescent signal when hybridized to the target RNA. Here we present our recent efforts to enhance the spatiotemporal resolution of RNA visualization by refining MBs.
We first asked if we could refine MBs to visualize defined subcellular populations of RNA in living neurons. To achieve this, we utilize visible light-activatable Q-dye MBs to allow only a subcellular fraction to be activated. Here, the fluorophore at the 5’-end was linked to a second quencher via a photolabile coumarin protecting group. Therefore, the MB only gives a fluorescent signal, when activated with visible light and hybridized to the target. This architecture allowed local activation of a hybridized subpopulation in a defined area of the cell. Knowing the exact origin of the activated RNA, we were able to increase the available monitoring time for neuronal mRNA from several minutes (literature known MBs) to more than 14 hours.
We next asked if it would be possible to gain spatiotemporal control over where the MB hybridization events occur. Therefore, we developed photo-tethered MBs where two phosphates in the loop backbone are covalently linked to each other via two photocages. This prevents the MB from hybridization to the target RNA. Only when light is applied, the photo-tethers are cleaved, and the inherent hybridization function of the MB is activated. This architecture allowed us to control the hybridization of photo-tethered MBs in primary cultured neurons.
To this day, stroke is the leading cause of death and disability worldwide. Due to increasing age of the world population and poor lifestyle, the incidence is further rising. Besides mechanical thrombectomy as a surgical option, there is a lack of therapeutic options with recombinant tissue plasminogen activator (rt-PA) being the only approved drug for treatment for ischemic stroke. However, there are various problems that make the administration of rt-PA difficult. In particular, it can only be given for ischemic (not hemorrhagic) stroke, and there is a narrow time frame of 4.5 hours after onset of stroke, in which it can be successfully applied. While the success rates of combined thrombectomy with rt-PA are around 60%, less than 5% of patients receive this therapy.
ß-Hydroxybutyrate (BHB) is a ketone body that is formed in high amounts during fasting and lipolysis. Ketone bodes and the ketogenic diet have been shown to have neuroprotective properties in neurodegenerative diseases. In prior work of our group, the ketogenic diet was shown to have beneficial effects in mice after transient ischemia. In the present work, a single dose of BHB was tested for beneficial effects. For this purpose, microdialysis was used to demonstrate that BHB can cross the blood-brain barrier. For the next series of experiments, transient cerebral ischemia was induced in mice for 90 minutes by unilaterally occluding the middle cerebral artery (MCAO) with a silicone-covered filament. Behavioral tests one day after BHB administration showed that the moderate dose of 30 mg/kg, given immediately after reperfusion, improved the neurological score significantly whereas a lower (10 mg/kg) and a higher dose (100 mg/kg) had no effects The main part of the experiments focused on mitochondrial respiration as a potential mechanism of action for BHB. In isolated mitochondria from mouse brain, BHB (1-10 mM) was able to stimulate mitochondrial respiration stronger than pyruvate, but not as strong as succinate.. In the following experiments, MCAO was induced in vivo, and mitochondria were isolated and investigated ex vivo. Experiments were conducted 60 minutes, 24 hours, 72 hours, and 7 days after cerebral ischemia and reperfusion. Besides mitochondrial respiration (normalized to mitochondrial protein content or citrate synthase activity), several other parameters were monitored: the development of bodyweight throughout the experiment, citrate synthase activity, plasma metabolites and behavior to assess motor functions. Three behavioral tests were conducted: first, the Corner test, an experiment for measuring the extent of unilateral movement. Here, if a stroked mouse is put into a narrow corner (30°), it is most likely to turn unilaterally to the right, whereas an unimpaired mouse will turn to both sides randomly. From a total of 10 turns, a laterality index was calculated. Second, in the Chimney test, the mouse walks heads first into a tube. Once it reaches the end, the tube is tipped 90 degrees to stand on the table vertically. Motorically impaired animals have difficulties crawling backwards up to the top of the tube. The experiment was stopped if an animal did not reach the top of the tube within 60 seconds. Third, in the Rotarod test, the mouse is placed on a rotating beam on which it is supposed to walk for at least 60 seconds, and the time when the animal falls off the rotating tube is measured.
All animals that had undergone ischemia showed massive weight loss until 72 hours after reperfusion. Weight loss then stagnated and there was a trend of increasing weight 7 days after reperfusion. The behavioral analysis showed that 24 hours after reperfusion, BHB-treated animals performed significantly better in the Corner test, meaning their moving patterns were more heterogeneous than those of saline-treated animals and in the Chimney test. 72 hours after reperfusion, BHB-treated animals still performed significantly better in the Chimney test, but 7 days after reperfusion, the performances of BHB- and saline-treated animals were no longer different from each other in any of the behavioral tests. In separate experiments, the plasma metabolites glucose, lactate, and pyruvate were changed in the animals that had undergone ischemia but were not affected by BHB administration.
Mitochondrial respiration was tested at four time points after the administration of BHB after reperfusion – 60 minutes, 24 hours, 72 hours, and 7 days after transient cerebral ischemia. 60 minutes later, data showed an increase of oxygen consumption of the complexes I and II. OxPhos was also increased but the effect at this point, did not reach statistical significance. 24 hours after reperfusion, this effect was consolidated: complex I, complex II and OxPhos respiration were significantly improved in the BHB-treated group compared to saline...
Intrinsische und extrinsische Faktoren wie die Darreichungsform, Komedikation und genetische Polymorphismen können einen signifikanten Einfluss auf die Exposition des Wirkstoffes haben und in der Folge zu Veränderungen in der Wirksamkeit oder Sicherheit eines Wirkstoffes führen. Die Fähigkeit die Auswirkungen solcher Faktoren auf die Exposition und die pharmakologische Aktivität eines Wirkstoffes zu quantifizieren und zu extrapolieren, repräsentiert einen Meilenstein bei der Bestimmung der erforderlichen Dosisanpassungen und der Umsetzung von Risikomanagementstrategien in der klinischen Pharmakologie. Unter dem Blickwinkel der modellbasierten Arzneimittelforschung und -entwicklung (engl. model-informed drug discovery and development (MID3)) können dynamisch mechanistische Modelle, wie z. B. whole-body PBPK/PD-Modelle, für die Vorhersage des Effekts sowie der Wechselwirkung mehrerer Faktoren auf PK und PD nützlich sein und könnten daher als Orientierung für die Wahl der Formulierung und für klinische Dosierungsempfehlungen dienen.
Obwohl PBPK-Modelle in der Pharmabranche inzwischen routinemäßig zur internen Entscheidungsfindung und zur Unterstützung der regulatorischen Bewertung eingesetzt werden, bleibt das Vertrauen Waiver von speziellen klinischen pharmakologischen Studien für biopharmazeutische Anwendungen durch PBPK- Modellanalysen zu stützen eher gering. Andererseits hat sich die virtuelle Bioäquivalenz im Zusammenhang mit der Simulation klinischer Studien als ein vielversprechendes, aber noch unterentwickeltes Feld erwiesen, mit dessen Hilfe der Anwendungsbereich der PBPK-Modellierung in der Biopharmazeutik erweitert werden kann. So werden beispielsweise BCS-basierte Biowaiver für Wirkstoffe der BCS-Klassen II und IV derzeit von den Gesundheitsbehörden nicht akzeptiert. In einigen Fällen hat die PBPK-Modellierung durch Verknüpfung der In-vitro-Freisetzung mit der In-vivo-Performance der Formulierung jedoch gezeigt, dass ein solcher Ansatz unter Umständen wissenschaftlich gerechtfertigt sein könnte. Auf ähnliche Weise können PBPK-Modellierung und VBE verwendet werden, um klinisch relevante Spezifikationen für die Wirkstofffreisetzung festzulegen und den "safe space" der Freisetzung zu definieren (oder zu erweitern). Doch selbst bei Wirkstoffen, die Unterschiede im Umfang und in der Rate der Absorption außerhalb der Bioäquivalenzgrenzen aufweisen, was bedeutet, dass sie nicht als bioäquivalent und damit austauschbar angesehen werden können, kann die therapeutische Äquivalenz beibehalten werden, sofern dies durch eine Expositions-Wirkungs-Analyse und/oder eine Expositions-Sicherheits-Analyse unter Verwendung empirischer, halb- oder vollmechanistischer PK/PD-Modelle angemessen begründet wird.
Wie bereits erwähnt bieten PK/PD- und insbesondere PBPK/PD-Modelle einen mechanistischen Ansatz, der die Gewebekonzentrationen am Wirkort des Wirkstoffes mit der pharmakologischen Wirkung verknüpft. Im Rahmen dieser Arbeit wird zunächst ein Überblick über bestehende PK/PD-Modelle und deren mathematischen Umsetzung vorgestellt. Darüber hinaus sind wirkstoffspezifische Fallbeispiele mit einer offensichtlichen Entkopplung von PK und PD von besonderem Interesse, bei denen Expositionsschwankungen weniger kritisch, wenn nicht gar irrelevant für die pharmakologische Reaktion sind (Publikation 1).
In diesem Zusammenhang bietet PBPK Modellierung und Simulation die Möglichkeit die oben genannten wissenschaftlichen Überlegungen zu untersuchen, ungetestete Szenarios zu erforschen und schließlich evidenzbasiert und arzneimittelspezifische Empfehlungen für Bioäquivalenzprüfungen zu erteilen. Daher bestand das Hauptziel darin PBPK/PD-Modelle zu entwicklen, zu validieren und anzuwenden sowie virtuelle Trials zu simulieren, um den relativen Effekt der In-vitro/ In-vivo-Freisetzung, PK-Charakteristiken (z.b. die Halbwertszeit) und die intraindividuelle Variabilität bei der In-vivo-Arnzeimittelwirkung von BCS Klasse II schwach sauren Verbindungen zu beurteilen und einen PBPK-IVIVE integrierten Arbeitsablauf vorzuschlagen, um virtuelle Bioäquivalenzstudien durchzuführen.
Es wurden drei BCS Klasse II schwach saure Wirkstoffe (Naproxen, Flurbiprofen, Ibuprofen) mit ähnlicher Disposition und ähnlichen metabolischen Eigenschaften zur Untersuchung ausgewählt. Allgemein sind alle drei Wirkstoffe stark an Plasmaproteine gebunden und haben daher ein niedriges Verteilungsvolumen, niedrigen First-Pass-Effekt, niedrige systemische Clearance und eine nahezu vollständige Bioverfügbarkeit (F>0.9). Allerdings unterscheiden sie sich signifikant in ihrer Halbwertszeit: Für Naproxen beträgt t1/2≃20-24 h, für Flurbiprofen t1/2≃7 h und für Ibuprofen t1/2≃2 h, was moderate bis lange, moderate und kurze Halbwertszeiten widerspiegelt.
Für alle drei Wirkstoffe wurde ein systematischer Arbeitsablauf erstellt einschließlich: i) Charakterisierung von in vitro biopharmazeutischen Eigenschaften (z.b. Löslichkeit, Freisetzung) gefolgt von modellbasierten Analysen von In-vitro-Ergebnissen, ii) Entwicklung und umfassende Validierung von PBPK/PD-Modellen und iii) Simulierung und Risikoeinschätzung von Bioäquivalenzstudien. Die Fallstudien von Naproxen (Publikation 2) und Ibuprofen (Publikation 3) konzentrieren sich auf bewährte Verfahren der IVIVE für biopharmazeutische Parameter, Risikoabschätzung und Simulation von Bioäquivalenzstudien mit PBPK-Modellen, welche die inter-occasion Variabilität miteinbeziehen. Das Beispiel von Flurbiprofen (Publikation 4) hebt die Wichtigkeit des Verständnisses des relativen Einflusses von intrinsischen (z.b. genetische Polymorphismen) und extrinsischen (z.b. Komedikationen) Faktoren auf die PK und PD des Wirkstoffes hervor, wenn Empfehlungen für die Bioäquivalenz und die therapeutische Gleichwertigkeit gemacht werden. Alle drei Fallbeispiele liefern mechanistische Erkenntnisse über die Freisetzungssgrenzen, die für die In-vivo-Arneimittelwirksamkeit kritisch ist, unter Berücksichtigung der PK-Eigenschaften des Wirkstoffes und der physiologischen Variabilität mit dem Ziel den Status quo des aktuellen BCS-basierten Biowaiveransatzes in Frage zu stellen und integrierte In-vitro-, In-vivo- und In-silico-Paradigma der Risikobewertung für Waiver von In-vivo-Bioäquivalenzstudien einzuführen.
In dem letzten Teil der Arbeit werden Herausforderungen, Kenntnislücken und Möglichkeiten von PBPK/PD-Modellierung zur Unterstützung von Waivern von in vivo klinischen Studien im Bereich von oralen Biopharmazeutika diskutiert (Publikation 5).
Im Großen und Ganzen schlägt diese Dissertation biorelevante In-vitro-Methoden für die Vorhersage von In-vivo-Formulierungsperformance und neue PBPK/PD-Methoden vor, um Daten von in vitro biopharmazeutischen Experimenten zu den In-vivo-Bedingungen zu extrapolieren. Außerdem ist dies das erste Mal nach unserem Kenntnisstand, dass PBPK/PD-Ansätze zur Durchführung virtueller Bioäquivalenzstudien vorgeschlagen werden, die auch die inter-occasion Variabilität der Pharmakokinetik berücksichtigen. Desweiteren hebt diese Arbeit die Bedeutung von pharmakokinetischen Eigenschaften auf Bioäquivalenz-Ergebnissen hervor und stellt ein neues Konzept zur Risikoeinschätzung von Bioäquivalenz vor, in welchem die Bewertung des Bedarfs eines Waivers von einer In-vivo-Bioäquivalenzstudie sowohl auf biopharmazeutischen als auch pharmakokinetischen Wirkstoffeigenschaften basiert und quantitativ mit PBPK/PD-Modellierung bewertet wird.
Macroautophagy, herein referred to as autophagy, is an evolutionarily conserved homeostatic process that normally occurs inside eukaryotic cells which involves degradation of cytoplasmic substances via lysosomes. It can be induced by various conditions such as starvation and drug exposure, as well as be inhibited by numerous compounds. Under normal conditions, the doublemembrane autophagosomes engulf the cytosolic substrates and deliver them to lysosomes for digestion. These substrates include unnecessary or dysfunctional cell components, such as faulty macromolecules, organelles and even invading pathogens. Autophagosomes are formed through the co-operative work of various autophagy-related (ATG) proteins organized into complexes. Upon closure of the autophagosomes, they fuse with the acidic lysosomes, resulting in formation of autolysosomes and the delivery of lysosomal hydrolases to degrade the engulfed contents. The fusion of the autophagosome with lysosome is carried out by specific SNARE proteins, small GTPases and their effectors including tethers, adaptors and motor proteins. Autophagy is impaired in many human diseases including cancer, neurodegenerative diseases, aging and inflammation. Therefore, manipulation of autophagy pathway holds a great promise for new therapeutic applications ...
Zika-virus (ZIKV), a flavivirus mainly transmitted by Aedes mosquitoes, is a single-stranded, positive-sense RNA virus. The viral genome is surrounded by a nucleocapsid and a lipid bilayer, in which membrane and envelope proteins are embedded. ZIKV disease is mainly characterized by mild symptoms, such as fever, rash as well as pain in head and joints. However, after epidemics it caused in the Americas in 2015/16, ZIKV infections were also associated with severe neurological complications like the Guillain-Barré syndrome (GBS) and microcephaly in fetuses and newborns. So far there are no specific antiviral treatments or vaccines available against ZIKV. This strengthens the need for a detailed understanding of the viral life cycle and virus-host interactions.
The antiviral host factor tetherin (THN) is an interferon-stimulated protein and therefore part of the cellular innate immune response. It comprises an N-terminal cytoplasmic domain, followed by a transmembrane helix, an extracellular coiled-coil domain and a C-terminal glycosylphosphatidylinositol (GPI) anchor. Containing two sites for membrane insertion linked by a flexible structure, THN is able to integrate into the membrane of budding viruses, thereby attaching them to each other and to the cell membrane and preventing their further release and spread.
In this study, the crosstalk of ZIKV and THN was analyzed. Previous gene expression analyses by microarray and quantitative polymerase chain reaction (qPCR) had revealed a strong upregulation of the BST2 gene encoding for THN in ZIKV-infected cells. However, this enhanced expression did not correlate with an enhanced THN protein level. On the contrary, the amount of THN in THN-overexpressing cells was after infection even heavily reduced. Furthermore, immunofluorescence analyses revealed a loss of THN membrane localization in these cells. By performing a cycloheximide assay, this loss could be traced back to a reduced protein half-life of THN in infected versus uninfected cells. Treatment with inhibitors of different protein degradation pathways as well as colocalization analyses with markers of several subcellular compartments indicated an involvement of the endo-lysosomal route. A knock-down of the ESCRT-0 protein HRS however prevented the sorting of THN for lysosomal degradation and led to a stabilization of THN protein levels. After HRS depletion, the release and spread of viral particles was reduced in THN-overexpressing compared to wildtype cells.
Taken together, the data obtained in this study revealed the potential of THN to restrict ZIKV release and spread. The enhanced degradation of THN in ZIKV-infected cells via the endo-lysosomal pathway could therefore be explained as an effective viral escape strategy. This could be circumvented by knockdown of the ESCRT-0 protein HRS, which highlighted HRS as a potential target for the development of antiviral treatments.
Pulsed dipolar (PD) EPR spectroscopy is an established and reliable tool for the investigation of biomolecules. In terms of long distance and orientation measurements, it is one of the leading methods and further fields of application are constantly being explored. The distances that can be detected with PD EPR also correspond to the range in which almost all important biomolecule interactions occur. In the transition from in vitro spectroscopy to in-cell spectroscopy, the power of PD EPR spectroscopy is particularly evident. It is non-invasive, more sensitive than NMR, and does not exhibit background signals from diamagnetic molecules. In particular, the absence of background signals is of great importance given the high density of molecules within cellular environment. However, like any other spectroscopic method, PD EPR has certain limitations. Owing to the intrinsically fast electron spin echo dephasing at higher temperature, these experiments are commonly carried out in frozen solutions at about 50 K. This temperature is far away from the physiological conditions and the freezing additives used, e.g. glycols, can further influence the structure. To enable measurements with and within living organisms, it is therefore necessary to ascend from the cold depths of the frozen state. At the same time, one has to adapt the spin tags for the desired application. Established nitroxides commonly used for EPR studies are typically susceptible to reduction. Thus, for studies under physiological conditions, e.g. in the cell, one has to fight against the reductive environment in the cell and somehow protect the spin labels. Initial published in-cell experiments within the research group and investigations of homogeneously distributed labeled double-stranded (ds) ‐DNA samples in solid matrices showed promising results and enabled pulsed measurement in the temperature range of 50‐ 295 K. It could also be demonstrated that spherical shielded nitroxides have a significantly longer life span in cellular environments than non-protected ones and first nuclear acids were measured in cell. Based on these results, we have gone further to overcome the standing limitations and developed the use of PD EPR spectroscopy. This work addresses these challenges with the overall goal of advancing the applications of PD EPR spectroscopy for studying biomolecules under physiological conditions.
We have focused on four different approaches. The results of these studies were published in various publications. They are presented and discussed together with further studies and put into the context of research conducted before and after the authors' publications.
In approach 1, we fought against the two main obstacles for using pulsed dipolar spectroscopy at ambient conditions – minimizing phase memory time T2 and averaging of the anisotropic dipolar coupling by rotational diffusion. We focused on an immobilization approach, while using rigid spin labels at same time. Besidesto the distance information, the incorporated rigid spin labels will give additional angular constrains and information about the molecular dynamics.
In approach 2, we focused on the on-site and on-demand formation of nitroxide spin labels using light-sensitive alkyl protection groups. This a very mild and efficient procedure that will hardly interfere with sensitive functional groups present in oligonucleotides or peptides. By establishing this method and using coumarin protecting groups plus two-photon excitation, this property may offer the potential to generate spin labels with very high levels of spatial and temporal resolution.
For approach 3, we used paramagnetic Gd3+ -ions as intrinsically stable labels, which are not reducible within a cellular environment. Easy to mix and bound to encodable lanthanide binding tags within the molecule Interleucin 1β, we were able to measure distances between two tags with PELDOR spectroscopy. We tested the extent to which this system is suitable for in-cell measurements.
Finally, we focus on methods for easier labeling by using non-covalentlabeling techniques. One of these is the novel nitroxide G´ for site-directed spin labeling of nucleic acids, especially for RNA. This spin label is sterically hindered, easy to build and binding occurs in seconds by simply mixing the spin label with the target. For large RNAs, another easy-to-mix and noncovalent spin-labeling strategy will be experimentally accompanied and presented.
The approaches and results described here are intended to demonstrate that the study of the biological functions of biomolecules under physiological conditions by pulsed EPR spectroscopy is feasible and operational. In combination, they will enable the life sciences to make further and faster progress in the search for the molecular master plan.
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in early childhood. Despite recent advances in the treatment regimes of rhabdomyosarcoma, the 5-year survival is still alarmingly low for the more aggressive metastasizing alveolar rhabdomyosarcoma subtype. Novel treatment strategies are needed in order to increase the overall survival rate. Hallmarks of cancer include evade cell death induction and evade immune system surveillance. This is mediated in part by up-regulation of inhibitor of apoptosis (IAP) proteins. With the development of Smac mimetic compounds mimicking the endogenous IAP antagonist Smac, this tumor evasion mechanism became exploitable.
In this PhD thesis, a combinatory approach for a putative treatment option of RMS will be presented. Here, the Smac mimetic compound BV6 will be used as a pre-treatment of RMS cells. This leads to a sensitizing effect within the tumor cells, increasing the killing efficacy of natural killer (NK) cells.
Subtoxic concentrations of BV6 were chosen to sensitize RMS cells. To remodel the solid tumor characteristics of RMS, a multicellular RMS tumor spheroid culture model was used.
In both tumor spheroids and conventional monolayer cell culture BV6 induced the degradation of IAP proteins (cIAP1, cIAP2, in spheroids XIAP). Further, BV6 led to the activation of both, the canonical and non-canonical NF-κB signaling pathways.
This was demonstrated by an increased IκBα and p65 phosphorylation, and nuclear translocation of p-p65, indicative for an active canonical NF-κB signaling. On the other side, cIAP degradation led to the stabilization and accumulation of NIK and downstream partial degradation of p100 to p52 and its nuclear translocation, indicating non-canonical NF-κB signaling pathway activity. A bulk RNA sequencing approach of BV6 treated RH30 cells validated the NF-κB signaling involvement and identified 182 differentially expressed genes. Among the interesting target genes are NFKBIA (IκBα),BIRC3 (cIAP2), NFKB2 (p100), CCL5 and SSTR2. SSTR2 was thoroughly validated as being up-regulated on a transcriptional and on protein level. Here, SSTR2A, one of the two alternative splicing variants, is up-regulated and opens a hypothetical targeted treatment strategy, as SSTR2 expression is not associated with RMS, but rather described with neuroendocrine tumor entities. In addition, CCL5 was thoroughly validated as a BV6 induced target. Again, the up-regulated mRNA transcription was validated by an increased translation and by increased secretion of CCL5. As CCL5 being associated as pro-migratory and activating of NK cells, CRISPR/Cas9 mediated CCL5 knock-out studies were performed to evaluate the influence of CCL5 within a BV6 pre-treatment and NK cell co-cultivation setting. It was shown that CCL5 knock-out does not rescue BV6 pre-treated RMS spheroids from NK cell attack and killing.
The previous mentioned transcriptional activity by BV6 stimulation was NIK mediated as knock-down of NIK reduced the mRNA transcription of several interesting genes.
However, NIK mediated down-stream signaling had no influence on the BV6 induced sensitizing effect towards NK cell mediated attack. A NIK knock-down had no rescue effect upon BV6 pre-treatment and NK cell co-treatment.
As cIAP proteins are present in receptor bound complexes, e.g. complex I at the TNF receptor 1 (TNFR1), a putative involvement of death receptors in general was evaluated.
Indeed, BV6 treatment of RMS cells could increase the surface presentation of DR5, a death receptor ligating TRAIL. Functionally, co-treatment of BV6 with TRAIL led to an additive cell death inducting effect. However, within the NK cell co-cultivation setting, addition of a neutralizing TRAIL anitbody could not rescue BV6 pre-treated RMS spheroids from NK cell killing. A similar effect was observed when neutralizing TNFα by adding Enbrel during the NK cell co-cultivation. BV6 sensitization of RMS spheroids seems to be independent of death receptors.
In addition to activating NF-κB, BV6 as a Smac mimetic is supposed to be able to release caspases bound by IAP proteins. Indeed, BV6 pre-treatment of RMS spheroids and co-cultivation with NK cells could cleave and thereby activate the executioner caspase-3. Further, treatment with a pan-caspase inhibitor, zVAD.fmk, could reduce the BV6 mediated sensitizing effect towards NK cell attack in RD spheroids.
Taken together, BV6 does induce a thoroughly validated NF-κB signaling pathway, leading to a NIK mediated transcriptional signature change. However, the NF-κB activation might not be responsible for the observed sensitization. Further, BV6 in combination with NK cells led to a seemingly death receptor independent, caspase dependent cell death induction of RMS spheroids. Although the mechanism remains partially con-cealed, a therapeutic benefit by combining a cell death sensitizing compound, i.e. BV6, with cytotoxic lymphocytes is evident.
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Vari-ous studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXAlo group displayed a high expression of IRX1 and the HOXAhi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXAlo/IRX1hi group was characterized to possess a strongly ele-vated relapse incidence compared to the HOXAhi/IRX1lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leuke-mia” aimed to investigate a potential correlation between the HOXAlo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
Meat adulteration is a global problem which undermines market fairness and harms people with allergies or certain religious beliefs. In this study, a novel framework in which a one-dimensional convolutional neural network (1DCNN) serves as a backbone and a random forest regressor (RFR) serves as a regressor, named 1DCNN-RFR, is proposed for the quantitative detection of beef adulterated with pork using electronic nose (E-nose) data. The 1DCNN backbone extracted a sufficient number of features from a multichannel input matrix converted from the raw E-nose data. The RFR improved the regression performance due to its strong prediction ability. The effectiveness of the 1DCNN-RFR framework was verified by comparing it with four other models (support vector regression model (SVR), RFR, backpropagation neural network (BPNN), and 1DCNN). The proposed 1DCNN-RFR framework performed best in the quantitative detection of beef adulterated with pork. This study indicated that the proposed 1DCNN-RFR framework could be used as an effective tool for the quantitative detection of meat adulteration.
The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5′ end, the ribosomal frameshift segment and the 3′-untranslated region (3′-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
Leukemia is a cancer of the blood and bone marrow characterized by an uncontrolled proliferation and accumulation of abnormal white blood cells. Leukemia can be classified based on the course of the disease (acute or chronic) and the blood cell type involved (myeloid or lymphocytic), leading to four main subtypes: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML). Leukemia represents 2.5% of all new cancer cases per year, and survival rates in some leukemias remain low at 40%.
The bone marrow microenvironment (BMM) is a system within the bone marrow comprising cellular and acellular components, all of which play a major role in hematopoiesis, providing the physical space where hematopoietic stem cells (HSCs) reside. The BMM interacts with HSCs, offering a “niche” for those cells and in case of leukemia, the BMM has a supportive role in disease maintenance and progression by supporting Leukemia stem cells (LSCs). One of the components of the BMM are calcium ions. Calcium is the most abundant mineral in the body, a key component of bones and is released by parathyroid hormone (PTH) induced bone remodeling. Calcium ions play a role in the localization, engraftment and adhesion of normal HSC to extracellular matrix (ECM) proteins in the BMM via the calcium sensing receptor (CaSR), thereby maintaining normal hematopoiesis. In addition of a major regulator of calcium homeostasis, CaSR contribute to the development of different cancers, functioning as either tumor suppressor or oncogene, depending on the involved tissue. However, the role of CaSR and its associated pathways in the local BMM for the development of leukemia is poorly understood. We hypothesized that calcium ions released from bone, subject to a fine balance between osteoblasts and osteoclasts, and/or CaSR, contribute to development, progression and response to therapy.
We have shown that the local calcium concentration forms a gradient in the bone marrow niche and in mice with CML is similarly low as in control mice, but significantly higher in mice suffering from BCR ABL1 driven B ALL or MLL AF9 driven AML. Similarly, the calcium concentration in the human BMM was found to be higher in AML than in other leukemias. Regarding the function of calcium in leukemia cells, we found that AML and CML cells respond differently to calcium exposure, with AML cells exhibiting regulation of cellular processes such as adhesion to the ECM protein fibronectin and migration toward CXCL 12, whereas CML cells remained mostly unaltered. Using genetic deletion or overexpression of CaSR in murine models of leukemia, we observed that CaSR acts as tumor suppressor in BCR-ABL1 driven CML and B ALL and as oncogene in AML.
Focusing on AML, our data shows that deficiency of CaSR on LICs leads, on one hand to increased apoptosis, and on the other hand to reduced cell cycle, reactive oxygen species (ROS) production and DNA damage in vivo, which may explain the observed prolongation of survival of mice. Complementary, in vitro experiments demonstrated that cells overexpressing CaSR have a distinct, cancer promoting phenotype compared to wildtype cells. Overexpression of CaSR led to an increase in proliferation, cell cycle, ROS production, DNA damage and reduced apoptosis. We have identified CaSR mediated pathways in AML and shown that CaSR enhances leukemia progression by activating MAPK/ERK and Wnt β catenin signaling. In addition, the CaSR interacting protein filamin A (FLNA) was shown to contribute to aggressive disease in vitro and in vivo. Furthermore, the mechanism underlying the role of CaSR in AML pathogenesis and possible regulation of LSCs was studied. Our findings demonstrated that CaSR ablation reduces myeloid progenitor function and proved that CaSR is required for maintenance of LSC pool by regulating its frequency and function. Further supporting the role of CaSR in LSC maintenance, genes associated with AML stemness and self renewal capacity were upregulated when CaSR was overexpressed and downregulated when CaSR was depleted. Given the role of CaSR in AML, the CaSR antagonist NPS 2143 was tested in vivo. The combination treatment of NPS 2143 with the standard of care, ara C, significantly reduced the tumor burden and prolonged the survival of mice with AML in syngeneic and xenotransplantation experiments. Based on the finding that CaSR functions as a tumor suppressor in CML, treatment of mice with the CaSR agonist cinacalcet in combination with imatinib prolonged survival of mice with CML compared to treatment with the mice given vehicle.
Our results suggest that calcium ions stemming from the calcium-rich BMM via CaSR strongly and differentially influence leukemia progression. As an adjunct to existing treatment therapies, targeting of CaSR with specific pharmacologic antagonists may prolong survival of patients with AML.
In dieser Arbeit konnte 1,8-Diborylnaphthalin (11) präparativ in einer Stufe und 65% Ausbeute aus dem literaturbekannten Boronsäureanhydrid 9 dargestellt werden. 11 ist das zweite bekannte, aromatisch verbrückte Derivat des Diborans B2H6. 11 kann als Startverbindung für eine Reihe strukturverwandter BNB-dotierter Phenalenderivate verwendet werden. Dazu werden zwei der vier Bor-gebundenen Protonen durch die Umsetzung mit einem Mesitylgrignard und Trimethylsilylchlorid substituiert. Die Umsetzung mit Wasser bzw. Aminen liefert BOB- bzw. BNB-Phenalene unter Freisetzung von elementarem Wasserstoff. Alle, auf diese Weise dargestellten Verbindungen, zeigen reversible Redoxeigenschaften und Photolumineszenz mit zum Teil besonders scharfen Emissionssignalen mit Halbhöhenbreiten von bis zu 31 nm. Zusätzlich wurden drei analoge Vertreter einer NBN-Phenalen Spezies dargestellt und charakterisiert. Die entgegengesetzte Dotierung äußert sich in einem grundlegend verschiedenem Redoxverhalten. Abschließend wurde die Reduktion des BNB-Phenalens 22 untersucht. Dabei gelang es das Radikal K[32] zu charakterisieren und seine Abbaureaktion in THF aufzuklären.
Biomoleküle, insbesondere Membranproteine (MPs), sind oftmals sehr sensitiv gegenüber ihrer chemischen Umgebung, wie pH-Wert, Puffer, Salzkonzentration und vielen weiteren Faktoren. MPs stabil und funktional in Lösung zu halten ist nicht trivial. Sie stellen deshalb eine besondere Herausforderung bei der Analyse von biologischen Systemen dar. Aus diesem Grund wurden und werden nach wie vor sogenannte membrane mimicking-(MM-) Systeme, wie beispielsweise Nanodiscs (NDs) oder styrene-maleic acid lipid particles (SMALPs), untersucht und entwickelt, um MPs eine naturähnliche Umgebung in Form einer Lipid-Doppelschicht zu bieten und sie so in ihrer natürlichen Konformation und natürlichen Funktionsweise/Aktivität in Lösung zu halten.
Laser induced liquid bead ion desorption (LILBID) Massenspektrometrie (MS) hat sich als hervorragende analytische Methode herausgestellt, um MPs in Kombination mit MM-Systemen zu untersuchen. LILBID-MS bietet nicht nur die Möglichkeit Proteine an sich zu identifizieren, sondern ermöglicht ebenfalls eine zerstörungsfreie Analyse von nicht-kovalent gebundenen Proteinkomplexen, sowie die Detektion einzelner Subkomplexe eines Proteinkomplexes. Auch die Analyse von Protein-Ligand-Wechselwirkungen ist möglich. Bei der LILBID-Ionisationsmethode werden kleine Tröpfchen erzeugt, die einen wässrig gelösten Analyt enthalten. Die Analyt-Tröpfchen werden anschließend mittels IR-Laser bestrahlt, wodurch der Analyt freigesetzt und massenspektrometrisch analysiert werden kann.
Diese Dissertation beschäftigt sich zum einen mit der Analyse des Lyse-Proteins ΦX174-E der Bakteriophage ΦX174, zum anderen mit Untersuchungen zur Histidinkinase SpaK aus B. subtilis in Kombination mit MMs. Weiterhin wird die Frage geklärt, ob und wie gut sich LILBID-MS zur Analyse von Saposin-Nanopartikel-(SapNPs)-solubilisierten MPs eignet. Darüber hinaus wird in dieser Dissertation die Darstellung von SapNP-solubilisierten MPs mittels zellfreier Proteinsynthese näher charakterisiert und untersucht welche Parameter aus präparativer Sicht optimiert werden können.
In vorausgegangenen Analysen von ND-solubilisierten MPs mittels LILBID-MS zeigte sich, dass manche in Verbindung mit NDs genutzten Lipide unerwünschte Signale im Spektrum zur Folge haben, die aus massiven Lipid-Anhaftungen am MSP oder dem Analyten resultieren. Überlappungen der m/z-Signale verschiedener Analyt- und/oder Komplexkomponenten mit diesen Lipid-Cluster-Signalen kann wiederum zum Verlust von Informationen führen. Daher beschäftigt sich ein weiterer Teil dieser Arbeit mit der Frage, ob durch den Einsatz von UV-schaltbaren Lipiden der Anwendungsbereich und/oder die Auflösung von LILBID-MS erweitert und verbessert werden kann.
Um biologische Prozesse zu verstehen ist es ebenfalls wichtig die zeitlichen/kinetischen Aspekte einer Reaktion zu untersuchen/kennen, sowie molekulare Prozesse gezielt zu kontrollieren. Licht hat sich hierbei als ein hervorragendes Werkzeug in der Analytik, sowie in der molekularen Prozesskontrolle etabliert. Licht bietet den Vorteil sehr selektiv eingesetzt werden zu können und sowohl orts- als auch zeitaufgelöst Informationen liefern zu können. Das gezielte Triggern einer Reaktion oder einer Protein-Protein-Interaktion kann beispielsweise durch sog. photo-cleaving von photolabilen Schutzgruppen ermöglicht werden. Bisweilen bietet die native MS nur wenig Möglichkeiten schnelle Reaktionen zu analysieren und kinetische Informationen zu gewinnen. Daher beschäftigt sich ein weiterer Teil dieser Dissertation damit zu untersuchen, ob und wie sich lichtgesteuerte Reaktionen im LILBID-Ionisationsprozess induzieren und gegebenenfalls auch zeitlich analysieren und charakterisieren lassen können.
Oxidative stress is thought to be a driver for several diseases. However, many data to support this concept were obtained by the addition of extracellular H2O2 to cells. This does not reflect the dynamics of intracellular redox modifications. Cells actively control their redox-state, and increased formation of ROS is a response to cellular stress situations such as chronic inflammation.
In this study, it was shown that different types of ROS lead to different metabolic and transcriptomic responses of HUVECs. While 300 μM extracellular H2O2 led to substantial metabolic and transcriptomic changes, the effects of DAO-derived H2O2 and menadione were low to moderate, indicating that the source and the concentration of ROS are important in eliciting changes in metabolism and gene expression.
Specifically, it was identified that acute increases in ROS transiently inactivate the enzyme ω-amidase/NIT2 of the glutaminase II pathway, which supplies cells with anaplerotic α-ketoglutarate. The pathway has not been studied systematically because, as noted above, the major intermediate, KGM, is not commercially available. In the present study, an internal standard for targeted detection of KGM in cells and blood plasma/serum was used. Deletion of NIT2 by CRISPR/Cas9 significantly reduced α-ketoglutarate levels in HUVECs and elevated KGM levels. It appears that in cell culture conditions, hydrolysis of KGM to α-ketoglutarate is very efficient. Knockout of the glutamine transaminases significantly reduced methionine, suggesting that the glutaminase II pathway is an important source of amino acid replenishment.
Similar to genetic silencing of GLS1 [91,92], HUVECs lacking NIT2 showed reduced proliferation and angiogenic sprouting. Furthermore, our results indicate that, at least in HUVECs, the enzyme also locates in the mitochondria where it interacts with key enzymes of glutamine/glutamate/α-ketoglutarate metabolism.
The data of the present work indicate that the glutaminase II pathway is an underappreciated, redox-sensitive pathway for glutamine utilization in HUVECs. Genetic deletion of NIT2 has considerable physiological effects highlighting the importance of glutamine for ECs.
Damit in der Schule die Vermittlung eines adäquaten Energieverständnisses gelingen kann, benötigt es eine Lehrkräfteausbildung, die dessen Herausforderungen in den Blick nimmt und die angehenden (Chemie-) Lehrerinnen und Lehrer aus fachwissenschaftlicher und didaktischer Perspektive vorbereitet. Denn in die Unterrichtsvorbereitung fließen neben bildungspolitischen und curricularen Vorgaben auch die Vorstellungen und Überzeugungen der Lehrkräfte mit ein. Zu den Herausforderungen, mit denen Lernende wie Lehrende konfrontiert sind, zählen die verschiedenen mentalen Repräsentationen zum Wort Energie aus Alltag und Naturwissenschaft, die zahlreichen chemischen Fachkontexte, in denen Energie bzw. Energiephänomene eine Rolle spielen, die unterschiedlichen Wissensnetze, die mit dem Begriff in den verschiedenen Naturwissenschaften verknüpft sind und der Einfluss der Fach- bzw. Alltagssprache.
Die (angehenden) Lehrkräfte fühlen sich auf diese Aufgabe oftmals fachlich nicht ausreichend vorbereitet. Um die Lehrkräfteausbildung in ihrem ersten Ausbildungsabschnitt auf die genannten Herausforderungen anzupassen und Lehrformate zu erweitern, benötigt es umfangreiche Kenntnisse über die mentalen Repräsentationen der Studierenden zur Energie sowie die damit verbundenen alternativen Konzepte zu schulrelevanten und lehrplanorientierten Themenschwerpunkten und die sprachlichen Besonderheiten. Die Vielschichtigkeit des Begriffs Energie erfordert eine ganzheitliche Betrachtung aller Aspekte, die es so bislang nicht gibt.
Aus diesem Grund ist es Ziel dieser Studie, die mentalen Repräsentationen der Studierenden, wie auch deren alternative Konzepte zu ausgewählten energiebezogenen Fachbegriffen aus den Bereichen chemische Bindungen, Thermodynamik und chemische Reaktionen zu erheben, in einen gemeinsamen fachlichen und sprachlichen Kontext zu setzen und daraus Rückschlüsse auf das Energieverständnis zu ziehen.
Im Sinne des Modells der didaktischen Rekonstruktion wird eine fachliche Klärung zum Untersuchungsgegenstand Energie durchgeführt. Für die Erhebung der empirischen Daten findet ein Rückgriff auf halbstandardisierte Leitfadeninterviews statt. Zielgruppe sind angehende Chemielehrkräfte, die mindestens im 5. Fachsemester Chemie für das Lehramt an Gymnasien studierten. Die Auswertung der Interviews erfolgt unter Rückgriff auf die qualitative Inhaltsanalyse nach Mayring und wird mit quantifizierenden Elementen trianguliert.
Die Studie zeigt die Erklärungsvielfalt des Begriffs Energie auf, denen sich die Studierenden bedienen. Dabei werden vor allem Beispiele einzelner Energiephänomene oder Energieformen herangezogen. In den verschiedenen Fachkontexten konnten diverse alternative Konzepte detektiert werden. Darüber hinaus konnten übergreifende Herausforderungen detektiert werden. Erkennen die Studierenden Widersprüche in ihrem Energieverständnis, wird Energie als abstrakt und schwer fassbar beschrieben. Zudem wird eine anthropozentrische Sicht eingenommen. Die angehenden Lehrkräfte neigen zu einer starken Kompartmentalisierung und begründen Wissenslücken mit der Zugehörigkeit zu anderen Fachwissenschaften. Eine weitere wichtige Erkenntnis aus der Studie ist, dass in den Fachwissenschaftlichen Veranstaltung die qualitativen Diskussionen angeregt werden müssen. Die zukünftigen Lehrerinnen und Lehrer bewegen sich in einem Spannungsverhältnis zwischen Fachwissenschaft und Didaktik und sind sich dessen sehr deutlich bewusst, indem sie bei Begriffsdefinitionen und Erklärungen die Anschaulichkeit der Exaktheit vorziehen. Es besteht die Notwendigkeit, Fachbegriffe in einem größeren Zusammenhang zu erläutern und die Studierenden zur Kommunikation darüber anzuregen.
This work deals with the theoretical investigation of the vibrationally promoted electronic resonance (VIPER) experiment, the intramolecular energy transfer within a rhodamine-BODIPY antenna system initiated by two-photon excitation and a computational study of the photochemical mechanism of the uncaging of the [7-(dimethylamino)coumarin-4-yl]methyl (DEACM) class of photocages . In continuation to Jan von Cosel’s work, the setup for the theoretical investigation of the VIPER experiment has been extended to two-photon absorption (TPA) also including the first-order Herzberg-Teller (HT) effects which are dependent on changes with respect to nuclear coordinates.
The VIPER experiment constitutes an extended form of two-dimensional infrared (2DIR) spectroscopy with a sequence of infrared (IR) and ultraviolet (UV) or visible (vis) pulses. The molecular system under probe is excited initially by a narrow-band IR pump pulse and then electronically excited by an off-resonant UV/vis pulse. An IR probe pulse is applied afterwards to probe the system and record a 2DIR spectrum in combination with the first pulse. Since the lifetime of the vibrational excitation is very short, the electronic excitation by the UV/vis pulse is used to enlarge the lifetime of the excitation in the molecule and thus enable measurements on a longer timescale. Therefore, it becomes easier to study dynamical photochemical processes on long timescales. In the VIPER experiment with TPA, the UV/vis pulse is replaced by a near-infrared (NIR) pulse which offers an intrinsic 3D resolution, minimzed photodamage, a lower noise level and an increased penetration depth. This makes TPA highly attractive for biological systems among a wide range of other possible applications.
The computation of the vibrationally resolved electronic absorption spectra accounts for the Franck-Condon (FC) contributions which are independent of the nuclear framework as well as the HT effects which are dependent on the nuclear coordinates. The FC contributions are dominant for electronically-allowed transitions whereas HT contributions could be important for weakly-allowed or forbidden transitions. Laying emphasis on TPA, the test systems used belong to the category of two-photon active compounds. The initial candidate is dimethylaminonitrodibenzofuran (DMA-NDBF) which has been reported to be a two-photon only caging compound. The other system is a well-known laser dye, a rhodamine derivative of the commercially available rhodamine 101 (Rh101). Rhodamines are also recognized for their excellent TPA characteristics.
The findings for both the test systems show interesting contrasts. The one-photon absorption (OPA) and TPA spectrum together with vibronic couplings present the same lineshape in case of DMA- NDBF and also the HT effects have very weak contributions to the vibronic spectrum. Insignificant HT effects are quite typical for electronically allowed transitions. Overall, the NO2 bending mode exhibits the strongest change in the absorption spectrum upon vibrational pre-excitation, even stronger than in the case of different ring distortion modes that usually show a high VIPER activity. In the case of rhodamine, the vibronic OPA spectrum is pre-dominantly the FC spectrum and the HT couplings have a very weak contribution. The vibronic TPA spectrum is entirely dominated by the HT contributions and hence, the vibrationally resolved TPA spectrum of the rhodamine is a HT-only spectrum. Explanations towards this behaviour have been reported by Milojevich et al. which are holding the change in symmetry of the molecular orbital transitions from the ground to the excited state accountable. No significantly VIPER-active normal modes could be determined owing to the low magnitudes of their dimensionless displacements that are connected to the Huang-Rhys factors. Two ring distortion modes however have been probed but the intensity of their vibrational pre-excitation is observed to be very low.
The other part of this work is concerned with the estimation of the rate of the intramolecular energy transfer within rhodamine-BODIPY dyads. After the investigations on the prospective rhodamine derivatives, the Rho101 derivative shows the highest TPA activity. This linked together with the BODIPY derivative with styryl substituents through an acetylene bond has been probed theoretically as well as experimentally for the excitation energy transfer (EET).
Time-resolved spectroscopic measurements reveal an ultrafast energy transfer process on femtosecond timescales. The theoretical estimation of the EET rates through the Förster theory and the determination of the coupling between the donor and acceptor groups by the transition density cube (TDC) method falls short of the experimental results. Because of this disagreement, quantum dynamics simulations with the multi-layer multi-configuration time-dependent Hartree (ML-MCTDH) method have been performed on an adapted rhodamine-BODIPY molecular dyad which reveal that the energy transfer occurs through transient coherence whose mechanism cannot be described by Förster theory ...
Die vorliegende Dissertationsarbeit behandelt eine umfangreiche Studie des nukleären Rezeptors (NR) TLX (engl. tailless homolog, TLX). Als ligandenaktivierbarer Transkriptionsfaktor ist TLX in Differenzierungs- und Proliferationsprozessen involviert und übernimmt somit eine tragende regulatorische Rolle in der Neurogenese von neuronalen Stammzellen87,88. Zahlreiche Studien haben gezeigt, dass eine fehlgesteuerte TLX-Expression mit gravierenden kognitiven, visuellen und neurodegenerativen sowie tumorigenen Erkrankungen assoziiert ist, sodass TLX ein vielversprechendes Wirkstofftarget mit hohem therapeutischem Potential darstellt94,95,99,100 105. Die pharmakologische Validierung von TLX als neues Wirkstofftarget befindet sich allerdings aufgrund limitierter Verfügbarkeit von validierten und potenten synthetischen und natürlichen kleinen organischen Molekülen in einer frühen Phase. Daher ist das Interesse sehr groß neuartige und wünschenswerterweise selektive TLX-Modulatoren zu generieren109,119-121.
Im Rahmen dieser Dissertationsarbeit wurden zu diesem Zweck mehrere Reportergenassays eingeführt, die die in vitro Aktivitätsstudie von TLX sowohl im Gal4-Hybridformat in Kombination mit Gal4-VP16 als starken Transkriptionsaktivator als auch als TLX-Volllängenprotein in HEK293T-Zellen (engl. human embryonic kidney, HEK293T) erlaubten. Zusätzlich wurde Gal4-TLX in Kombination mit VP16-RXRα untersucht, um bisherige unbekannte potentielle Heterodimer-vermittelte Effekte zu studieren. In einem primären Screeningansatz im Gal4-Format unter Verwendung einer kommerziell erhältlichen Wirkstofffragmentbibliothek und ausgewählter strukturähnlicher Wirkstoffe wurden mehrere Wirkstofffragmentkandidaten identifiziert (30, 34, 39, 45 und 47), die einen attraktiven Ausgangspunkt zur Darstellung von TLX-Modulatoren darstellten. Insgesamt wurden in vier Projekten vier strukturdiverse Chemotypen anhand von Struktur-Wirkungs-Beziehungs-Studien anhand der Aktivität an TLX untersucht. Ausgehend von Fragment 34 beinhaltete das erste Projekt die Identifizierung und Charakterisierung von Xanthinderivaten als inverse TLX-Agonisten. Eine systematische Struktur-Wirkungs-Beziehungs-Studie lieferte mehrere hochpotente Derivate, die auf das Grundgerüst von 8-Phenyltheophyllin (97) basierten. Parallel konnte Istradefyllin (116), welches aktuell zur Behandlung der Parkinson-Erkrankung in den USA und Japan Anwendung findet, als potenter inverser TLX-Agonist identifiziert werden. Mehrere orthogonale zelluläre und zellfreie Experimente klassifizierten die Xanthine als neue erste TLX-Modulatoren. Das zweite Projekt umfasste die Identifizierung und Charakterisierung des unselektiven β-Adrenorezeptorblockers Propranolol (54) ausgehend vom Wirkstofffragment 30. Durch eine vorläufige systematische Struktur-Wirkungs-Beziehungs-Untersuchung der aliphatischen Aminoalkoholseitenkette von 54 konnte die sekundäre Aminogruppe als determinierendes Strukturmotiv für eine Aktivität an TLX bestimmt werden. Weitere Migrations- und Zellviabilitätsexperimente demonstrierten erste phänotypische Effekte in T98G-Glioblastomzellen seitens 54, die TLX-vermittelt sein könnten. Das dritte Projekt behandelte die Darstellung eines potenten neuartigen TLX-Agonisten mit Hilfe eines ligandenbasierten Pharmakophormodells. Das verwendete Pharmakophormodell wurde hierbei unter Verwendung des publizierten Referenzliganden ccrp2 (2) und dem identifizierten Wirkstofffragment 45 aus dem vorherigen Screeningansatz generiert. Durch eine anschließende rationale Fragmentfusion von 45 mit weiteren TLX-Agonisten aus dem Wirkstofffragmentscreening konnte der neuartige potente TLX-Agonist 137h synthetisiert werden, welcher eine verbesserte mikrosomale Stabilität im Vergleich zu 45 und 2 aufwies. Das vierte Projekt beinhaltete die Darstellung neuartiger TLX-Modulatoren mit Hilfe eines Scaffold Hopping Ansatzes. Hierbei wurden essentielle Strukturmotive aus der Xanthin-Struktur-Wirkungs-Beziehung (erstes Projekt) auf weitere Wirkstofffragmente übertragen. Die Validierung dieses Scaffold Hoppings anhand der Verbindung 156 führte anhand eines darauf folgenden kombinatorisch-chemischen Ansatzes zur Darstellung einer Substanzbibliothek (255 Amidrohprodukte). Ein Aktivitätsscreening der Amidrohprodukte deutete in den Reportergenassays auf drei aktive TLX-Modulatoren hin (582, 611 und 629), welche nachträglich gezielt synthetisiert, isoliert und erneut auf Aktivität an TLX validiert wurden. Hierbei hob sich besonders 629 hervor, welches in drei orthogonalen zellulären Reportergenassays TLX-vermittelte Effekte aufwies und zusätzlich einen Bindungseffekt an rekombinanter exprimierter TLX-Ligandenbindedomäne zeigte.
Mit dieser Arbeit konnte mit Hilfe der Einführung diverser TLX-basierter Reportergenassays zur Aktivitätsstudie von TLX mehrere strukturdiverse Liganden als potentielle tool compounds identifiziert und charakterisiert werden. Alle abgeleiteten TLX-Modulatoren können somit als wertvolle neue Startpunkte zur Derivatisierung neuartiger potenter Liganden und somit zu einem Fortschritt in der pharmakologischen Validierung von TLX als Wirkstofftarget dienen.
Despite a high clinical need for the treatment of colorectal carcinoma (CRC) as the second leading cause of cancer-related deaths, targeted therapies are still limited. The multifunctional enzyme Transglutaminase 2 (TGM2), which harbors transamidation and GTPase activity, has been implicated in the development and progression of different types of human cancers. However, the mechanism and role of TGM2 in colorectal cancer are poorly understood. Here, we present TGM2 as a promising drug target.
In primary patient material of CRC patients, we detected an increased expression and enzymatic activity of TGM2 in colon cancer tissue in comparison to matched normal colon mucosa cells. The genetic ablation of TGM2 in CRC cell lines using shRNAs or CRISPR/Cas9 inhibited cell expansion and tumorsphere formation. In vivo, tumor initiation and growth were reduced upon genetic knockdown of TGM2 in xenotransplantations. TGM2 ablation led to the induction of Caspase-3-driven apoptosis in CRC cells. Functional rescue experiments with TGM2 variants revealed that the transamidation activity is critical for the pro-survival function of TGM2. Transcriptomic and protein–protein interaction analyses applying various methods including super-resolution and time-lapse microscopy showed that TGM2 directly binds to the tumor suppressor p53, leading to its inactivation and escape of apoptosis induction.
We demonstrate here that TGM2 is an essential survival factor in CRC, highlighting the therapeutic potential of TGM2 inhibitors in CRC patients with high TGM2 expression. The inactivation of p53 by TGM2 binding indicates a general anti-apoptotic function, which may be relevant in cancers beyond CRC.
Fluorescently labeled nanoparticles are widely used for evaluating their distribution in the biological environment. However, dye leakage can lead to misinterpretations of the nanoparticles’ biodistribution. To better understand the interactions of dyes and nanoparticles and their biological environment, we explored PLGA nanoparticles labeled with four widely used dyes encapsulated (coumarin 6, rhodamine 123, DiI) or bound covalently to the polymer (Cy5.5.). The DiI label was stable in both aqueous and lipophilic environments, whereas the quick release of coumarin 6 was observed in model media containing albumin (42%) or liposomes (62%), which could be explained by the different affinity of these dyes to the polymer and lipophilic structures and which we also confirmed by computational modeling (log PDPPC/PLGA: DiI—2.3, Cou6—0.7). The importance of these factors was demonstrated by in vivo neuroimaging (ICON) of the rat retina using double-labeled Cy5.5/Cou6-nanoparticles: encapsulated Cou6 quickly leaked into the tissue, whereas the stably bound Cy.5.5 label remained associated with the vessels. This observation is a good example of the possible misinterpretation of imaging results because the coumarin 6 distribution creates the impression that nanoparticles effectively crossed the blood–retina barrier, whereas in fact no signal from the core material was found beyond the blood vessels.
The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1–D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical β2-β3-β5 interfaces. Evidently, the 3′-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.
Copper perchlorophthalocyanine (CuPcCl16, CuC32N8Cl16, Pigment Green 7) is one of the commercially most important green pigments. The compound is a nanocrystalline fully insoluble powder. Its crystal structure was first addressed by electron diffraction in 1972 [Uyeda et al. (1972). J. Appl. Phys. 43, 5181–5189]. Despite the commercial importance of the compound, the crystal structure remained undetermined until now. Using a special vacuum sublimation technique, micron-sized crystals could be obtained. Three-dimensional electron diffraction (3D ED) data were collected in two ways: (i) in static geometry using a combined stage-tilt/beam-tilt collection scheme and (ii) in continuous rotation mode. Both types of data allowed the crystal structure to be solved by direct methods. The structure was refined kinematically with anisotropic displacement parameters for all atoms. Due to the pronounced crystal mosaicity, a dynamic refinement was not feasible. The unit-cell parameters were verified by Rietveld refinement from powder X-ray diffraction data. The crystal structure was validated by many-body dispersion density functional theory (DFT) calculations. CuPcCl16 crystallizes in the space group C2/m (Z = 2), with the molecules arranged in layers. The structure agrees with that proposed in 1972.
Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase β (IKKβ). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.
Acute myeloid leukemia (AML) is one of the most frequently occurring and fatal types of leukemia. Initiated by genetic alterations in hematopoietic stem and progenitor cells, rapidly proliferating cancer cells (leukemic blasts) infiltrate the bone marrow and damage healthy hematopoiesis. Subgroups of AML are defined by underlying molecular and cytogenetic abnormalities, which are decisive for treatment and prognosis. For AML patients that can be intensively treated, the first line treatment remains a combination of cytarabine and anthracycline, which was developed in the 1970s. While this treatment regimen clears the disease and reinstates normal hematopoiesis (complete remission, CR) in 60% to 80% of patients below the age of 60, CR rates in patients above the age of 60 are only 40% to 50%. Relapse and refractory disease are the major cause of death of AML patients, despite large efforts to improve risk-adjusted post-remission therapy with further chemotherapy cycles and, if possible, allogeneic bone marrow transplantation. Elderly patients are particularly difficult to treat because of age-related comorbidities and because their disease tends to relapse more often than the disease of younger patients. Thus, the cure rates of AML vary with age, with 5-year survival rates of about 50% in young patients, and less than 20% in patients above the age of 65 years. With the median age of AML patients being 68 years, the need for novel therapeutic options is immense. The recent approval of eight new agents (venetoclax, midostaurin, gilteritinib, glasdegib, ivosidenib, enasidenib, gemtuzumab ozogamicin and CPX-351 (liposomal cytarabine and daunorubicin)) has added considerably to the therapeutic armamentarium of AML and has increased cure rates in specific subgroups of AML. However, the high heterogeneity among patients, clonal evolution and commonly occurring drug resistance, which cause the high relapse rates, remain a substantial problem in the treatment of AML. Therefore, a better understanding of currently used therapeutics and further development of novel therapeutics is urgently needed.
In recent years, attention has increasingly focused on therapeutic strategies to interfere with the metabolic requirements of cancer cells. The last three decades have provided extensive insights into the diversity and flexibility of AML metabolism. AML cells use different sources of nutrients compared to normal hematopoietic progenitor cells and reprogram their metabolic pathways to fulfill their exquisite anabolic and energetic needs. As a result, they develop high metabolic plasticity that enables them to thrive in the bone marrow microenvironment, where oxygen and nutrient availability are subject to constant change.
Cancer cells, specifically AML cells, have a strong dependency for the amino acid glutamine. Glutamine serves in energy production, redox control, cell signaling as well as an important nitrogen source. The only enzyme capable of de novo glutamine synthesis is glutamine synthetase (GS). GS catalyzes glutamine production from glutamate and ammonium. In AML, the metabolic role and dependency of GS is poorly understood. Here, we investigated the effects of GS deletion on AML growth, and its functional relevance in AML metabolism. Genetic deletion of GS resulted in a significant decrease of cell growth in vitro, and impaired leukemia progression in vivo in a xenotransplantation mouse model. Interestingly, the dependency of AML cell growth on GS was shown to be independent of its functional role in glutamine synthesis. Glutamine starvation did not increase the dependency of the AML cells on GS, nor did increased glutamine availability rescue the GS-knockout-associated growth disadvantage. Instead, functional studies revealed the role of GS in the detoxification of ammonium. GS-deficient cells showed elevated ammonium secretion as well as a higher sensitivity towards the toxic metabolite. Exogenous provision of 15N-labeled ammonium was detoxified by GS-driven incorporation into glutamine. Studies on cells that had gained resistance to GS-knockout-mediated growth inhibition indicated enzymes involved in the urea cycle and the arginine biogenesis pathway to compensate for a loss of GS. Together, these findings unveiled GS as an important ammonium scavenger in AML.
Clinical studies on AML patients revealed increased ammonium concentrations in the blast-infiltrated bone marrow compared to peripheral blood. In line with this finding, proteome and transcriptome analysis of AML blasts showed a significant upregulation of GS in AML compared to healthy progenitors, further indicating its importance in ammonium detoxification.
Analyzing pathways that contribute to ammonium production revealed protein uptake followed by amino acid catabolism as a yet not identified mechanism supporting AML growth. Protein endocytosis and subsequent proteolytic degradation were shown to rescue AML cells from otherwise growth-inhibiting glucose or amino acid depletion. Furthermore, protein metabolization led to the reactivation of the mammalian target of rapamycin (mTOR) signaling pathway, which was deactivated upon leucine and glutamine depletion, revealing protein consumption as an important alternative source of amino acids in AML.
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The DNA damage response (DDR) is a vast network of molecules that preserves genome integrity and allow the faithful transmission of genetic information in human cells. While the usual response to the detection of DNA lesions in cells involves the control of cell-cycle checkpoints, repair proteins or apoptosis, alterations of the repair processes can lead to cellular dysfunction, diseases, or cancer. Besides, cancer patients with DDR alterations often show poor survival and chemoresistance. Despite the progress made in recent years in identifying genes and proteins involved in DDR and their roles in cellular physiology and pathology, the question of the involvement of DDR in metabolism remains unclear. It remains to study the metabolites associated with specific repair pathways or alterations and to investigate whether differences exist depending on cellular origin. The identification of DDR-related metabolic pathways and of the pathways that cause metabolic reprogramming in DDR-deficient cells may produce new targets for the development of new therapies.
In this thesis, nuclear magnetic resonance spectroscopy (NMR) was used to assess the metabolic consequence of the loss of two central DNA repair proteins with importance in diseases context, ATM and RNase H2, in haematological cells. An increase in intracellular taurine was found in RNase H2- and ATM-deficient cells compared to wild-type cells for these genes and in cells after exposition to a source of DNA damage. The rise in taurine does not appear to result from an increase in its biosynthesis from cysteine, but more likely from other cellular processes such as degradation pathways.
Overall, evidence for metabolic reprogramming in haematological cells with faults in DNA repair resulting from ATM or RNase H2 deficiencies or upon exposition to a source of DNA damage is presented in this study.
Famotidine inhibits toll-like receptor 3-mediated inflammatory signaling in SARS-CoV-2 infection
(2021)
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-κB pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19.
Chronic inflammation is characterized by persisting leukocyte infiltration of the affected tissue, which is enabled by activated endothelial cells (ECs). Chronic inflammatory diseases remain a major pharmacotherapeutic challenge, and thus the search for novel drugs and drug targets is an ongoing demand. We have identified the natural product vioprolide A (vioA) to exert anti-inflammatory actions in vivo and in ECs in vitro through inhibition of its cellular target nucleolar protein 14 (NOP14). VioA attenuated the infiltration of microglia and macrophages during laser-induced murine choroidal neovascularization and the leukocyte trafficking through the vascular endothelium in the murine cremaster muscle. Mechanistic studies revealed that vioA downregulates EC adhesion molecules and the tumor necrosis factor receptor (TNFR) 1 by decreasing the de novo protein synthesis in ECs. Most importantly, we found that inhibition of importin-dependent NF-ĸB p65 nuclear translocation is a crucial part of the action of vioA leading to reduced NF-ĸB promotor activity and inflammatory gene expression. Knockdown experiments revealed a causal link between the cellular target NOP14 and the anti-inflammatory action of vioA, classifying the natural product as unique drug lead for anti-inflammatory therapeutics.
Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection
(2021)
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
The vascular endothelium is a monolayer of endothelial cells that builds the inner lining of the blood vessels and constitutes a regulatory organ within the physiological system to sustain homeostasis. Endothelial cells participate in physiological processes including inflammation and angiogenesis. Dysregulation of these processes, however, can evoke or maintain pathological disorders, including cardiovascular and chronic inflammatory diseases or cancer. Although pathological inflammation and angiogenesis represent treatable conditions, current pharmacotherapeutic approaches are frequently not satisfying since their long-term application can evoke therapy resistance and thus reduced clinical efficacy. Consequently, there is an ongoing demand for the discovery of new therapeutic targets and drug leads. Considering that endothelial cells play a critical role in both angiogenesis and inflammation, the vascular endothelium represents a promising target for the treatment of diseases.
Vioprolide A is a secondary metabolite isolated from the myxobacterium Cystobacter violaceus Cb. vi35. Recently, vioprolide A was identified to interact with NOP14, a nucleolar protein involved in ribosome biogenesis. Ribosome biogenesis is an indispensable cellular event that ensures adequate homeostasis. Abnormal alterations in the ribosome biogenesis, referred to as ribosomopathies, however, can lead to an overall increase in the risk of developing cancer. Accordingly, several studies have outlined the involvement of NOP14 in cancer progression and metastasis, and vioprolide A has been demonstrated to exert anti-cancer effects in vitro. However, the impact of vioprolide A and NOP14 on the endothelium has been neglected so far, although endothelial cells are crucially involved in inflammation and angiogenesis under both physiological and pathological conditions.
In the present study, the effect of vioprolide A on inflammatory and angiogenic actions was analysed. In vivo, the laser-induced choroidal neovascularization (CNV) assay outlined a strong inhibitory effect of vioprolide A on both inflammation and angiogenesis. Furthermore, intravital microscopy of the cremaster muscle in mice revealed that vioprolide A strongly impaired the TNF-induced leukocyte-endothelial cell interaction in vivo.
In further experiments, the specific effect of vioprolide A on activation processes of primary human umbilical vein endothelial cells (HUVECs) was examined. According to the in vivo results, vioprolide A decreased the leukocyte-endothelial cell interaction in vitro through downregulating the cell surface expression and total protein expression of ICAM-1, VCAM-1 and E-selectin. Vioprolide A evoked its anti-inflammatory actions via a dual mechanism: On the one hand, the expression of pro-inflammatory proteins, including TNFR1 and cell adhesion molecules, was lowered through a general downregulation of de novo protein synthesis. The inhibition of de novo protein synthesis is most likely linked to the interaction with and inhibition of NOP14 by vioprolide A in HUVECs. On the other hand, the natural product prevented the nuclear translocation and promotor activity of the pro-inflammatory transcription factor NF-ĸB. Interestingly, most anti-inflammatory compounds that interfere with the NF-ĸB signaling pathway prevent NF-ĸB nuclear translocation through recovering or stabilizing the inhibitory IĸB proteins. Vioprolide A, however, decreased rather than stabilized the IĸB proteins and prevented NF-ĸB nuclear translocation through interfering with its importin-dependent nuclear import. By performing siRNA-mediated knockdown experiments, we evaluated the role of NOP14 in inflammatory processes in HUVECs and could establish a causal link between the anti-inflammatory actions of vioprolide A and the deletion of NOP14.
Besides exerting anti-inflammatory actions, we found that vioprolide A potently decreased the angiogenic key features proliferation, migration and sprouting of endothelial cells. Mechanistically, the natural product interfered with pro-angiogenic signaling pathways. Vioprolide A reduced the protein level of growth factor receptors, including VEGFR2, which is the most prominent receptor responsible for angiogenic signaling in endothelial cells. This effect was based on the general inhibition of de novo protein synthesis by the natural product. Downregulation of growth factor receptors impaired the activation of downstream signaling intermediates, including the MAPKs ERK, JNK and p38. To our surprise, however, activation of Akt, another downstream effector of VEGFR2, was increased rather than decreased. Furthermore, vioprolide A lowered the nuclear translocation of the transcriptional coactivator TAZ, which is regulated by the evolutionary conserved Hippo signaling pathway. Interestingly, however, and in contrast to NF-ĸB, TAZ nuclear translocation in mammalian cells seems to be independent of importins. In this context, we found that vioprolide A reduced both the protein level and nuclear localization of MAML1, which is needed to retain TAZ in the nucleus after its successful translocation.
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The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.
Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
Standard cancer therapy research targets tumor cells while not considering the damage on the tumor microenvironment (TME) and its associated implications in impairing therapy response. Employing patients-derived organoids (PDOs) and matched stroma cells or a novel murine preclinical rectal cancer model of local radiotherapy, it was demonstrated that tumor cells-derived IL-1α polarizes cancer-associated fibroblasts towards an inflammatory (iCAFs) phenotype. While numerous studies in different tumor entities highlighted the molecular heterogeneity of CAFs, so far there are no clear findings on their functional heterogeneity and relevance in therapy resistance and response. The present study molecularly characterized iCAFs subpopulation among RCA patients as well as the preclinical mouse model and importantly unraveled the detailed molecular mechanism underlying their contribution to impair therapy response. Mechanistically, iCAFs were demonstrated to be characterized by an upregulation of nitric oxide synthase (iNOS) which triggered accumulation of reactive nitrogen species (RNS) and subsequently an oxidative DNA damage response (DDR). Such a baseline IL-1α-driven DNA damage further sensitized iCAFs to a p53-mediated therapy induced senescence (TIS) causing extensive extracellular matrix (ECM) changes and induction of senescence associated secretory phenotype (SASP) that favored tumor progression and hindered tumor cell death. Moreover, iCAFs reversibility and repolarization into more quiescent like phenotype was demonstrated upon IL-1 signaling inhibition by anakinra, a recombinant IL-1 receptor antagonist (IL1RA). Accordingly, treating mice with anakinra or specific deletion of Il1r1 in CAFs sensitized stroma-rich resistant tumors to chemoradiotherapy (CRT). Similarly, targeting CAFs senescence by senotherapy (venetoclax chemical) or employing Trp53 deficient mice reverted therapy resistance among non-responsive tumors in vivo by reducing ECM deposition and consequently favoring CD8+ T cells intratumoral infiltration posttherapy. Importantly, rectal cancer patients that do not completely respond to neoadjuvant therapy displayed an iCAFs senescence program post-CRT. Moreover, these patients presented a baseline increased CAFs content, a dominant iCAFs signature that correlated with poorer disease-free survival (DFS) and a significantly reduced circulating IL1RA serum levels. While reduced pretherapeutic IL1RN gene expression predicted poor prognosis among RCA patients, IL1RA serum levels were associated with rs4251961 (T/C) single nucleotide polymorphism (SNP) in the IL1RN gene. Finally, functional validation assays revealed that conditioned media of PDOs drove inflammatory polarization of fibroblasts and consequently rendered them sensitive to RNS-mediated DNA damage and TIS. Collectively, the study highlighted a crucial and novel role of a CAFs subset, iCAFs, in therapy resistance among RCA patients, shedding light on their functional relevance by identifying IL-1 signaling as an appealing target for their repolarization and successful targeting. Therefore, it makes sense to combine the newly demonstrated and thoroughly proven therapeutic approach of targeting IL-1 signaling in combination with conventional CRT and possibly immunotherapy. This might have a major impact on RCA therapy and be of immense relevance for other stroma-rich tumors.
Sphingosin 1 Phosphat (S1P) ist ein wichtiger Lipidmediator, der über G Protein gekoppelte Rezeptoren und intrazelluläre Wirkungen vielfältige Wirkungen auslöst und eine Rolle bei der Lymphozytenzirkulation, der Erhaltung der endothelialen Barriere, bei Entzündungsprozessen und Tumorwachstum spielt. Die S1P Lyase (Sgpl1) katalysiert den irreversiblen Abbau von S1P und damit den letzten Schritt des Sphingolipidkatabolismus‘. Ein Fehlen der Sgpl1 bewirkt eine Akkumulation von S1P und anderen Sphingolipiden im Blut und Gewebe, was multiple Organschäden zur Folge hat. Menschen mit S1P Lyase Insuffizienz Syndrom (SPLIS) leiden insbesondere unter steroidresistentem nephrotischem Syndrom, Nebennierenrinden-insuffizienz und neurologischen Störungen. Weitere mögliche Symptome sind Lymphopenie, Hautveränderungen und Dyslipidämien. S1P Lyase defiziente Mäuse weisen sehr ähnliche Organschädigungen auf.
An Sgpl1 Knockoutmäusen war zuerst die massive Akkumulation nicht nur von Sphingolipiden, sondern auch von Cholesterin und Triglyceriden in Blut und Leber aufgefallen. Auch bei SPLIS Patienten wurde eine Hypercholesterinämie beobachtet. Um die Kreuzregulation des Sphingolipid- und Cholesterinmetabolismus besser zu verstehen, sollte die Rolle der Sgpl1 in der Leber, dem Hauptort des Lipidmetabolismus, untersucht werden. Hierzu sollte ein Mausmodell mit einem hepatozytenspezifischen Sgpl1 Knockout (Sgpl1HepKO) etabliert und charakterisiert werden. Dies wurde durch Kreuzen von Sgpl1fl/fl-Mäusen mit Mäusen, welche die Cre-Rekombinase unter dem Albuminpromoter exprimierten, erreicht. Die basale Charakterisierung zeigte, dass diese Mäuse im Gegensatz zu globalen Sgpl1 Knockoutmäusen sowohl im Alter von acht Wochen, als auch im Alter von acht Monaten einen unauffälligen Phänotyp aufwiesen. Das äußere Erscheinungsbild inklusive Leber und Körpergewicht, das Blutbild, die Leberenzyme sowie die Histologie der Leber waren unverändert. Die Analyse der Leberlipide mit Hilfe von Hochleistungsflüssigkeits-chromatographie gekoppelt mit einer Tandem Massenspektrometrie zeigte eine signifikante Akkumulation (≈1,5 2 fach) von S1P, Sphingosin und Ceramiden, aber nicht von Glucosylceramiden und Sphingomyelin in der Leber. Messungen im Plasma zeigten eine Erhöhung mehrerer Ceramide, während der S1P Spiegel normal war. Ferner zeigten Untersuchungen der Galle signifikant erhöhte Konzentrationen an S1P, Dihydro S1P und Glucosylceramiden, jedoch unveränderte Ceramide. Die Ergebnisse legen folgende Schlussfolgerungen nahe: 1. In der Leber kann mit Hilfe von Ceramidsynthasen akkumulierendes Sphingosin in Ceramide umgewandelt werden, welche anschließend ins Blut sezerniert und letztendlich vermutlich von anderen Zellen verstoffwechselt werden. Außerdem ist nicht ausgeschlossen, dass S1P ebenfalls ins Blut sezerniert und dort effektiv abgebaut wird, so dass die S1P Konzentration im Plasma unverändert bleibt. 2. S1P sowie Glucosylceramide werden an die Galle abgegeben und ausgeschieden. 3. Die Sgpl1 in der Leber ist nicht essentiell für die Regulation des Plasma S1Ps, was zuvor vermutet worden war
Eine Analyse der Sterole zeigte in Sgpl1HepKO Mäusen erhöhte Spiegel an Cholesterin und Desmosterol in der Leber. In Übereinstimmung mit der erhöhten Proteinexpression des low density lipoprotein (LDL ) Rezeptors und erniedrigten Konzentrationen des LDL Cholesterins im Plasma, deuten diese Daten auf eine erhöhte Aufnahme von LDL Cholesterin durch die Leber hin. Untersuchungen in der Leber sowie mit primären Hepatozyten zeigten im Gegensatz zu globalen Sgpl1 Knockoutmäusen keine Veränderungen der Peroxisomen-Proliferator-aktiviertem Rezeptor γ Expression. Weitere Gene mit zentraler Rolle wie der Liver X receptor oder die Fettsäuresynthase, waren ebenfalls nicht reguliert. Dieser im Vergleich zu globalen Sgpl1-Knockoutmäusen milde Phänotyp lässt sich durch die deutlich geringere Akkumulation von Sphingolipiden aufgrund der oben beschriebenen Kompensations-mechanismen in Sgpl1HepKO Mäusen erklären.
In weiteren Untersuchungen sollten die Auswirkungen einer Sgpl1-Defizienz an Fibroblasten untersucht werden. Hierzu standen embryonale Fibroblasten aus Sgpl1 Knockoutmäusen zur Verfügung (Sgpl1-/- MEFs). In einer Kooperation mit Dr. Janecke von der Universität Innsbruck standen außerdem humane Fibroblasten eines SPLIS Patienten zur Verfügung.
An Sgpl1-/- MEFs war zuvor eine gestörte Calciumhomöostase festgestellt worden, welche sich durch eine erhöhte zytosolische Calciumkonzentration und vermehrte Calciumspeicherung im Endoplasmatischen Retikulum und in Lysosomen auszeichnete. Die Plasmamembran-Calcium ATPase (PMCA) trägt an Fibroblasten entscheidend zur Regulation der zytosolischen Calciumkonzentration bei. Ihre Expression auf Proteinebene war jedoch in Sgpl1-/- MEFs nicht verändert. Im Rahmen dieser Arbeit wurde durch eine Immunfärbung erstmals festgestellt, dass die PMCA in Sgpl1-/- MEFs nicht vollständig an der Plasmamembran lokalisiert war. Dies könnte der Grund für die erhöhte zytosolische Calciumkonzentration in den Zellen sein. ...
Kurz zusammengefasst waren die Ziele dieser Arbeit, die in vivo Untersuchung einer Hyperhomocysteinämie und spezifischer diätetischer Mikronährstoffe im Kontext der Alzheimer-Erkrankung. Zu diesem Zweck wurden zwei Krankheitsmodelle in den Mäusen induziert. Zum einen wurde eine Alzheimer-ähnliche Pathologie genetisch simuliert durch den Einsatz des neuen AppNL-G-F knock-in Modells, das im Zuge dieser Arbeit auch weiter charakterisiert wurde. Zum anderen wurde eine chronische Hyperhomocysteinämie in den Tieren induziert via Langzeit-Fütterung einer Spezialdiät, die defizient an den Vitaminen B6, B12 und Folat war, was sich durch erhöhte Werte der Aminosäuren Homocystein und Homocysteinsäure in verschiedenen biologischen Matrices der Mäuse wie Serum, Urin und Hirngewebe, bemerkbar machte. Durch die Kombination der Krankheitsmodelle wurden sowohl Aspekte einer familiären Alzheimer-Erkrankung (verstärkter Amyloid-β-Anabolismus im knock-in Modell) als auch ein potentielles Charakteristikum der sporadischen Form der Krankheit (erhöhte Homocystein-Spiegel) simuliert. Auswirkungen des AppNL-G-F Genotyps, einer zusätzlichen Hyperhomocysteinämie und potentiell vorteilhafter, oder gar präventiv wirksamer Mikronährstoffe wurden dabei mit Hilfe von diversen Verhaltensversuchen und ergänzenden ex vivo Analysen bewertet.
Trotz massiver cerebraler Amyloidose war lediglich ein milder Einfluss auf die kognitive Leistungsfähigkeit der AppNL-G-F Tiere im Vergleich zur gleichaltrigen Wildtyp-Kontrolle detektierbar. Dies weist zum einen auf die Subtilität des Mausmodells hin und zum anderen befeuert es die kontroverse, häufig geführte Diskussion um die zentrale Bedeutung der „Amyloid-Hypothese“ im Rahmen der komplexen Alzheimer-Pathologie. Die kognitiven Fähigkeiten der entsprechenden Mäuse verschlechterten sich auch nicht bei gleichzeitig signifikant erhöhten Homocystein- und Homocysteinsäurespiegeln, d.h. die Hyperhomocysteinämie hat in diesem Modell für familiären Alzheimer nicht kausal zur Verschlimmerung der induzierten Pathologie beigetragen sowohl hinsichtlich der kognitiven Leistung in diversen Verhaltensversuchen als auch hinsichtlich dem Schweregrad der cerebralen Amyloidose.
Zur Hyperhomocysteinämie, vor allem aber auch zur Rolle bestimmter diätetischer Interventionen in dem Kontext, findet man eine heterogene, teilweise konträre Literatur vor, insbesondere im klinischen Kontext. Die untersuchten diätetischen Ansätze in dieser Arbeit, bestehend aus hochdosierten B-Vitaminen, mehrfach ungesättigten Fettsäuren, Betain und einer komplexeren Mikronährstoffkombination, zeigten ebenfalls keinen konsistenten Effekt auf Phänotyp und Amyloid-β-Menge in den Hirnen der Tiere. Die Ergebnisse der durchgeführten Studien legen daher, zumindest in diesem Krankheitsmodell, keinen Wert als potentiell präventiven Ansatz der kognitiven Verschlechterung bei Alzheimer nahe.
Da die dieser Arbeit zugrundeliegenden in vivo Studien keine per se erhöhten, AppNL-G-F-assoziierten Homocysteinspiegel offenbarten, zeigte sich Homocystein nicht als Biomarker, zumindest für die in diesem Mausmodell simulierten Aspekte der komplexen Alzheimer-Pathologie. Neben den zuvor beschriebenen fehlenden Effekten der Hyperhomocysteinämie, konnten in dieser Arbeit jedoch auch statistisch signifikante Einflüsse sichtbar gemacht werden. Wie in der durchgeführten Kinetikstudie gezeigt, resultierte die Alzheimer-ähnliche Pathologie in einem signifikant höheren Schweregrad der ausgebildeten Hyperhomocysteinämie in den AppNL-G-F Tieren im Vergleich zur gleichaltrigen Wildtyp-Kontrolle. Folglich übte der gestörte Amyloid-β-Metabolismus, neben der B-vitamindefizienten Diät, einen zusätzlich verstärkenden Effekt auf den hyperhomocysteinämischen Status aus. Sowohl für knock-in-, als auch Wildtyp-Tiere konnte gezeigt werden, dass bei Beendigung der Karenz an Vitamin B6, B12 und Folat, die erhöhten Homocystein- und Homocysteinsäurespiegel innerhalb kurzer Zeit wieder auf Baseline-Niveau normalisiert werden können.
Weitere signifikante Effekte wurden detektiert bezüglich Erythrozyten-bezogener Parameter wie den Hämoglobingehalt im Blut der hyperhomocysteinämischen Tiere. Ein reduzierter Sauerstofftransport und die damit einhergehende verringerte Versorgung der Neuronen mit Sauerstoff in den entsprechenden experimentellen Gruppen deuten auf eine vornehmlich vaskuläre Wirkung hin im Hinblick auf Homocystein-bezogene Pathomechanismen, die potentiell zu einer Demenz beitragen. Solche Effekte können zusätzlich verstärkt worden sein durch die, in der durchgeführten Proteomanalyse gezeigte, Herunterregulierung angiogener Marker im Serum und in der Cerebrospinalflüssigkeit dieser Tiere. Eine Verringerung der kapillären Dichte im Hirn und ein verringerter cerebraler Blutfluss haben ein zusätzlich reduziertes Angebot an Sauerstoff und Glucose zur Folge und stellen einen Link zu eingeschränkter kognitiver Leistungsfähigkeit in Älteren und Alzheimer-Patienten dar. Translational relevant ist eine vaskuläre Wirkung von Homocystein auch dadurch, dass vaskuläre Demenz und Alzheimer in etwa 40% der Fälle koinzident sind und Homocystein in früheren Humanuntersuchungen eine größere Bedeutung bei der vaskulären Demenz im Vergleich zur Alzheimer-Erkrankung nahelegte.
Auch wenn in Summe die beschriebenen Effekte der Hyperhomocysteinämie nicht groß genug waren, um sich in phänotypischen Einschränkungen in den Tieren auszudrücken, so konnten in der hier vorliegenden Arbeit dennoch Details zur Rolle erhöhter Homocysteinspiegel für verschiedene biologische Prozesse aufgeklärt werden. Insbesondere die Funde der explorativen Proteomanalyse in Serum und CSF könnten Ansatzpunkte für weitergehende Untersuchungen darstellen und sollten in anderen präklinischen Krankheitsmodellen und/oder einer Humanstudie validiert werden.
Additive manufacturing or 3D printing as an umbrella term for various materials processing methods has distinct advantages over many other processing methods, including the ability to generate highly complex shapes and designs. However, the performance of any produced part not only depends on the material used and its shape, but is also critically dependent on its surface properties. Important features, such as wetting or fouling, critically depend mainly on the immediate surface energy. To gain control over the surface chemistry post-processing modifications are generally necessary, since it′s not a feature of additive manufacturing. Here, we report on the use of initiator and catalyst-free photografting and photopolymerization for the hydrophilic modification of microfiber scaffolds obtained from hydrophobic medical-grade poly(ε-caprolactone) via melt-electrowriting. Contact angle measurements and Raman spectroscopy confirms the formation of a more hydrophilic coating of poly(2-hydroxyethyl methacrylate). Apart from surface modification, we also observe bulk polymerization, which is expected for this method, and currently limits the controllability of this procedure.
The stress-dependent dynamics of Saccharomyces cerevisiae tRNA and rRNA modification profiles
(2021)
RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H2O2, NaAsO2 or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA.
The prevalence and specificity of local protein synthesis during neuronal synaptic plasticity
(2021)
To supply proteins to their vast volume, neurons localize mRNAs and ribosomes in dendrites and axons. While local protein synthesis is required for synaptic plasticity, the abundance and distribution of ribosomes and nascent proteins near synapses remain elusive. Here, we quantified the occurrence of local translation and visualized the range of synapses supplied by nascent proteins during basal and plastic conditions. We detected dendritic ribosomes and nascent proteins at single-molecule resolution using DNA-PAINT and metabolic labeling. Both ribosomes and nascent proteins positively correlated with synapse density. Ribosomes were detected at ~85% of synapses with ~2 translational sites per synapse; ~50% of the nascent protein was detected near synapses. The amount of locally synthesized protein detected at a synapse correlated with its spontaneous Ca2+ activity. A multifold increase in synaptic nascent protein was evident following both local and global plasticity at respective scales, albeit with substantial heterogeneity between neighboring synapses.
Bezüglich der Arzneimittelforschung galt für sehr lange Zeit das Paradigma "ein Gen, ein Medikament, eine Krankheit". In jüngerer Zeit ändert sich dieses Paradigma jedoch auf Grund von redundanten Funktionen und alternativen sich kompensierenden Signalmustern, die insbesondere bei Krebserkrankungen vorherrschend sind. Daher kann die logische Konsequenz nur sein, Multi-Target-Strategien gegenüber Single-Target-Ansätzen in Betracht zu ziehen. Auf Grund der Schwierigkeit, mit einer Kombination von zwei Einzelwirkstoffen, in diesem Fall BET- und HDAC-Inhibitoren eine konsistente Biodistribution und Pharmakokinetik zu erreichen, wurde nach Einzelmolekülen gesucht, die mehrere inhibitorische Aktivitäten aufweisen. Dies wurde hier zunächst durch die einfache Konjugation von zwei unterschiedlichen Pharmakophoren erreicht.
Insgesamt wurden vier verschiedene Liganden dieses Typs synthetisiert und einer von ihnen, Verbindung 14, zeigte sehr vielversprechende Ergebnisse. 14 vereint den BET Inhibitor JQ1- mit dem HDAC Inhibitor CI994 und hat eine hemmende Wirkung sowohl gegen BRD4- als auch HDAC-Proteine wie durch DSF- und nanoBRET-Assay gezeigt werden konnte. Außerdem zeigten in vitro Assays in PDAC-Zellen, dass 14 ein noch potenterer dualer BET/HDAC-Inhibitor ist als die Kombination aus JQ1 und CI994. Während die Effekte von 14 auf das BETi-Antwortgen MYC denen von JQ1 ziemlich ähnlich sind, sind insbesondere die HDAC-inhibitorischen Effekte nachhaltiger und verstärkt, wahrscheinlich aufgrund einer längeren Verweildauer von 14 auf HDAC als dies bei CI994 der Fall ist. Dies ist durch das hohe Niveau der acetylierten Lysine von Histon H3 im Western Blot erkennbar. Dieses veränderte Expressionsverhalten hatte einen großen Einfluss auf das Zellwachstum und überleben in allen getesteten PDAC-Zelllinien. Hier wurde die Überlegenheit von 14 gegenüber der gleichzeitigen Behandlung der Zellen mit JQ1 und CI994 sehr deutlich. Wurden PDAC-Zellen mit dem dualen Inhibitor 14 behandelt, hatte dies ein geringeres Wachstum und Überleben der Krebszellen zur Folge als mit beiden ursprünglichen Molekülen, unabhängig davon, ob diese einzeln oder simultan verabreicht wurden. Außerdem wurde 14 mit Gemcitabin, einem gut verträglichen Chemotherapeutikum, kombiniert, dass bei PDAC allein nur eine begrenzte Aktivität aufweist. Es stellte sich heraus, dass die Reihenfolge, in der die Medikamente verabreicht werden, einen großen Einfluss auf die Effektivität hatte. Der durch 14 induzierte Stopp des Zellzyklus verhindert den Einbau von Gemcitabin in die DNA, wenn 14 vor oder gleichzeitig mit Gemcitabin verabreicht wird. Wenn jedoch die Behandlung mit 14 nach der Verabreichung von Gemcitabin folgt, wird der durch Gemcitabin induzierte S-Phasen-Arrest und Replikationsstress aufrechterhalten. Im Vergleich zu den meisten früheren Studien, die sich mit dualen BET/HDAC-Inhibitoren beschäftigten, ist dies eine große Verbesserung, da es bisher keinen signifikanten Unterschied zwischen der Verwendung eines dualen BET/HDAC-Inhibitors und der Kombination von zwei Einzelinhibitoren gab.
Als Proof of Concept unterstützten die Daten weitere Bemühungen zur Entwicklung zusätzlicher dualer BET/HDAC-Inhibitoren. Daher wurden zwei weitere Generationen dualer BET/HDAC Inhibitoren entwickelt, die jedoch bisher nicht an die Eigenschaften von 14 anknüpfen konnten. Vor allem die 3. Generation bietet jedoch Raum für Optimierungen, so dass hier möglicherweise noch ein potenter dualer Inhibitor zu finden ist. Sollte es in Zukunft einen zugelassenen dualen BET/HDAC-Inhibitor geben, ist es jedoch nicht unwahrscheinlich, dass keine der hier verwendet BET inhibierenden Strukturen verwendet werden, aber Struktur des HDAC inhibierenden Teils immer noch vergleichbar ist. Der Grund dafür ist, dass die HDAC Inhibitoren größtenteils relativ einfach aufgebaut. So lange das wichtigste, die zinkbindende Gruppe vorhanden ist, scheint der Linker sowie die Capping-Gruppe zweitranging zu sein. Die größere Herausforderung wird vermutlich die Suche nach dem passenden BET Inhibitor sein und die Wahlmöglichkeiten sind schon jetzt vielfältig.
Generell lässt sich sagen, dass die Idee der dualen BET/HDAC-Inhibitoren äußerst vielversprechend und es wert ist, weiter verfolgt zu werden. Dies liegt vor allem an den guten Testergebnissen, die mit Verbindung 14 erzielt wurden. Mit Hilfe dieser Art von Inhibitoren könnte es in Zukunft möglich sein, die Überlebensrate von PDAC-Patienten zu erhöhen, wenn nicht als alleiniges Medikament, so vielleicht als Zusatz zur Chemotherapie. Darüber hinaus scheint der Einsatz von dualen BET/HDAC-Inhibitoren nicht nur auf die Behandlung von PDAC beschränkt zu sein und kann auch bei anderen Krebsarten angewendet werden. NMC zum Beispiel ist ein ebenso seltener wie tödlicher Subtyp des schlecht differenzierten Plattenepithelkarzinoms und zeichnet sich durch eine Fusion des NUT-Gens mit BRD4 aus, wodurch es potenziell anfällig für eine BET-Inhibition ist. Tatsächlich zeigte 14 auch hier einen größeren positiven Effekt auf die getesteten NMC-Zellen als JQ1 oder CI994 und veranlasste die Zellen unter anderem zur Differenzierung. ...
Standard biorelevant media reflect the average gastrointestinal (GI) physiology in healthy volunteers. The use of biorelevant media in in vitro experiments has become an important strategy to predict drug behaviour in vivo and is often combined with in silico tools in order to simulate drug plasma profiles over time. In addition to the healthy population, the effects of disease state or co-administration of other drugs on plasma profiles must be considered to assure drug efficacy and safety. Thus, there is a need for a more accurate representation of the human GI physiology when it is altered by disease or co-administered drugs in in vitro dissolution experiments.
This thesis focused on the development of biorelevant media and dissolution tests reflecting GI physiology in circumstances where the gastric pH is elevated. Diseases linked to an elevated gastric pH are hypochlorhydria and achlorhydria, but these days treatment with acid-reducing agents (ARAs) is the single greatest cause of elevation in gastric pH. pH-dependent drug-drug interactions (DDIs) with ARAs are frequent, as the ARAs are used in a number of diseases using a variety of drugs. As the drugs currently on the market are often poorly soluble and ionisable, their dissolution is highly dependent on the pH of the GI tract, especially the gastric pH.
The thesis research consisted of several steps. In the first step, physiological changes in the human GI tract during the therapy with ARAs were identified. Parameters of the standard biorelevant gastric medium FaSSGF were adjusted to the identified changes to reflect the impact of ARA co-administration on the gastric physiology. The media aim to assess the potential extent of the ARA impact on gastric physiology by introducing biorelevant media pairs, ARA pH 4 and pH 6 media, of which one reflects a lesser, and the other a stronger impact of ARAs.
In the second step these ARA media were implemented in in vitro dissolution set-ups.
The dissolution of poorly soluble ionisable drugs was assessed using one-stage, two-stage and transfer model set-ups, as well as using a more evolved in vitro system TIM-1. Comparison of results from dissolution set-ups using the standard, low pH, gastric biorelevant medium FaSSGF (pH 1.6 or 2), and the same set-ups using ARA pH 4 and pH 6 media, shows a decrease in dissolution rate and extent for weakly basic compounds PSWB 001 and dipyridamole, and an increase in rate and extent of dissolution for the weakly acidic compound raltegravir potassium, when the gastric pH is elevated. Due to different physicochemical properties, the extent of the impact of physiological changes during ARA therapy (when either ARA pH 4 or pH 6 medium is selected) on dissolution varied among the model drugs. Thus, the bracketing approach, which considers a range of the possible ARA co-administration impact on drug dissolution, was confirmed to be best practice in assessing the impact of ARAs.
In the third step, dissolution data from in vitro experiments with ARA media was implemented into in silico models. The predictions using various in silico model approaches in Simcyp™ Simulator (minimal and full PBPK model, dissolution input using DRM and DLM) successfully bracketed in vivo data on drug administration during ARA therapy and correctly predicted an overall decrease in plasma concentration for the two model weakly basic compounds and an increase in plasma concertation for the model weakly acidic compound.
In all assessed scenarios, the ARA methods proved to be an essential part of evaluating and predicting the impact of ARAs on drug pharmacokinetics, and appropriately predicted the extent of a possible impact of ARAs on the drug plasma profiles. Thus, the ARA biorelevant media and dissolution tests were demonstrated to be valuable tools reflecting administration of drugs when the gastric pH is elevated and able to predict the impact of ARA therapy on drug administration.
The ability to evaluate the impact of human (patho) physioloy on drug behaviour in the gastrointestinal tract is of great importance, as the GI conditions play a significant role in drug release and absorption. Thus, there is great interest on the part of the pharmaceutical industry and regulatory agencies to develop best practices in this field, especially for pH-dependent DDIs. The media and dissolution tests developed in this thesis are biorelevant methods appropriate for evaluation of the impact of elevated gastric pH on drug efficacy and safety. Such methods, used as a risk assessment tool, in connection with evaluation of the efficacy window and potential toxicity, may help to increase confidence about decisions as to whether a pH-effect will occur and whether it is relevant or not, prior to conducting clinical studies. They may also enable changes in inclusion/exclusion criteria during recruiting for large-scale efficacy trials. In fact, the biopharmaceutic approach to drug development is becoming standard practice on a number of fronts, including metabolic DDIs, renal and hepatic insufficiency, powering decision-making process and possibly even waiving certain types of clinical studies.
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Bacteria are true artists of survival, which rapidly adapt to environmental changes like pH shifts, temperature changes and different salinities. Upon osmotic shock, bacteria are able to counteract the loss of water by the uptake of potassium ions. In many bacteria, this is accomplished by the major K+ uptake system KtrAB. The system consists of the K+-translocating channel subunit KtrB, which forms a dimer in the membrane, and the cytoplasmic regulatory RCK subunit KtrA, which binds non-covalently to KtrB as an octameric ring. This unique architecture differs strongly from other RCK-gated K+ channels like MthK or GsuK, in which covalently tethered cytoplasmic RCK domains regulate a single tetrameric pore. As a consequence, an adapted gating mechanism is required: The activation of KtrAB depends on the binding of ATP and Mg2+ to KtrA, while ADP binding at the same site results in inactivation, mediated by conformational rearrangements. However, it is still poorly understood how the nucleotides are exchanged and how the resulting conformational changes in KtrA control gating in KtrB is still poorly understood.
Here,I present a 2.5-Å cryo-EM structure of ADP-bound, inactive KtrAB, which for the first time resolves the N termini of both KtrBs. They are located at the interface of KtrA and KtrB, forming a strong interaction network with both subunits. In combination with functional and EPR data we show that the N termini, surrounded by a lipidic environment, play a crucial role in the activation of the KtrAB system. We are proposing an allosteric network, in which an interaction of the N termini with the membrane facilitates MgATP-triggered conformational changes, leading to the active, conductive state.
The phospholipid bilayers are the primary constituents of the membrane in living cells in which lipids are hold together in bilayer leaflets through a combination of different forces into the liquid crystalline (Lα) phase. Despite their thin fragile formations, the phospholipid bilayers are responsible for performing a variety of important tasks in the cells, some of which are carried out directly by the lipid bilayers and some by various integral proteins embedded within the bilayers. There have been continues efforts over the past decades to replicate the compound biophysical properties of living cell membranes in model lipid bilayers.
An important question remains unanswered: is it possible to replicate physical properties under “non-equilibrium” conditions as found in cell membranes in model lipid bilayers? In almost all previous studies, the model lipid bilayers were under static conditions – for instance, at zero lateral pressure. However, in living organisms, the cell membranes are involved in continuous (nonequilibrium) exchange and (or) transport of lipid species with the surrounding environment which consequently leads them to experience continuous lateral pressure variations. One suitable in vitro approach is to spatiotemporally control the model lipid bilayers over a time period during which they can be spatially stimulated at a level compatible to that found under in vivo conditions. This can be achieved with high spatiotemporal resolution by making lipids light-dependent through implementation of azobenzene photoswitch in their structures.
In this study, a specific azobenzene containing photolipid (AzoPC) is integrated into POPE:POPG bilayers (POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)) at ~14 mol% to construct a photo responsive model bilayers entitled as photoliposomes. Magic angle spinning solid-state NMR spectroscopy (MAS-NMR) at high field (850 MHz) is the measurement technique of choice by which it is possible to pursue the dynamics (fluidity) of the bulk lipids within the photoliposomes at atomistic resolution. It is shown that the AzoPCs undergo an efficient trans-to-cis isomerization (~85%) within the photoliposomes as the result of UV light absorption, and thermally relax back to the trans state during a period of ~65 h under the MAS measurement conditions. The order parameter measurements based on the C−H dipolar couplings reveal that the non-equilibrium cis-to-trans thermal isomerization impact of AzoPC on the fluidity of the bulk lipid is highly localized – the fluidity perturbations originate from specific order parameter changes in the middle section of the bulk lipid acyl chains. Further 1H NOESY measurements confirm the hypothesis that the azoswitch topologies in either cis and trans conformer of the photolipid is the key parameter in localized alteration of the C−H order parameters along the bulk lipid acyl chains.
Diacylglycerol kinase (DgkA) from E. coli is an enzyme responsible for the phosphorylation of diacylglycerol to phosphatidic acid, at the expense of adenosine triphosphate. Structurally, DgkA is a homo oligomer composed of three symmetric 14 kDa protomers, each of which has three transmembrane helices and one surface helix. Upon embedding within the photoliposomes, it is shown that DgkA enhances the AzoPC localization impact on the fluidity of the bulk lipids. In this regard, the results of a series of statistical simulations of lipid lateral diffusions along the bilayer leaflets in presence and absence of embedded proteins are accompanied with those of experimentally measured based upon which it is justified that membrane proteins markedly limit lipid lateral diffusions in the bilayers. In case of the DgkA proteo-liposomes with lipid-to-protein ratio of 50, it is estimated that the diffusion coefficient of lipids is above 2-fold lower compared to that of the protein free liposomes.
The cis-to-trans AzoPC isomerization and its following consequence in localized alteration of the bulk lipid fluidity is further investigated on the structural dynamics and enzymatic functionality of the embedded DgkA within the proteo-photoliposomes. It is revealed that DgkA structural dynamics are perturbated in a multi-scale, complex manner. The dynamics of residues located in different regions of DgkA changes with the light-induced AzoPC isomerization, but their time courses differ from residue to residue. For example, 29Ala, a residue on the hinge between the surface helix and membrane helix-1, exhibits the steepest time-dependent cross peak intensity changes in time-resolved NCA spectra. The impact of the lasting membrane fluidity perturbation on the enzymatic functionality of the embedded DgkA is subsequently measured which demonstrates a significant variation under cis- and trans-AzoPC conformations within the proteo-photoliposomes.
The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.
Chlorsilane stellen Schlüsselsubstanzen zur Herstellung von elementarem Silicium dar. Zum Beispiel ist HSiCl3 ein wichtiger Ausgangsstoff im Siemensprozess[1, 2]. Chlorsilane werden unter anderem zur Herstellung von Silikonen im Müller-Rochow-Prozess[3-5] verwendet. Bei beiden Prozessen werden Silylene[6-10] als Schlüsselmediate in den Reaktionen angenommen. So auch bei der Bildung von höheren Perchlorsilanen (Reaktion b), die nur in Form komplexer Polymergemischen erhalten werden. In der vorliegenden Dissertation wurde die Plausibilität eines auf Silylen basierenden Reaktionsmechanismus zur Bildung und Reaktivität von Chlorsilanen quantenchemisch untersucht.Mit den Berechnungen aus dieser Arbeit konnten diese molekularen Prozesse aufgeklärt werden[33-35].
Die quantenchemischen Rechnungen aus dieser Arbeit umfassen Kalibrierungsrechnungen an Chlorsilanen, um die Leistungsfähigkeit der quantenchemischen Methoden zu beurteilen.
Hierbei wurden berechnete Strukturen mit den experimentellen verglichen. Zusätzlich wurden Standardbildungsenthalpien berechnet, um diese auch mit den experimentellen Daten zu vergleichen. Nach Prüfung der Validität der Referenzmethoden wurde die Tragfähigkeit der rechengünstigeren Methoden der Dichtefunktionaltheorie evaluiert.
In Vereinbarung von Rechenaufwand und Genauigkeit einer Rechenmethode wurden thermochemische Stabilitäten, Reaktionsenergien zur Bildung von Chlorsilanen aus Schema 1 berechnet. Die Betrachtung der Reaktionsmechanismen erfolgte sowohl in der Gasphase als auch in Lösung. Dabei wurden die Bildung von cyclo-Chlorsilanen, kettenförmigen und verzweigten Chlorsilanen betrachtet. Unterstützend konnten alle Intermediate und Produkte unter Verwendung der ausgewählten quantenchemischen Methode mit 29Si-NMR-Rechnungen begleitet werden. Hierbei wurden auch Vergleichsdaten von nicht literaturbekannten 29Si-NMR Verschiebungen erstellt.
In the recent years, myxobacteria have emerged as a novel source of natural compounds with structural diversity and biological activity for drug discovery. In this work, the two myxobacterial compounds archazolid and vioprolide were characterized for their potential pharmacological effects in vascular endothelial cells. Archazolid is a wellestablished v-ATPase inhibitor found in Archangium gephyra and Cystobacter spec. As the v-ATPase represents a promising target in cancer treatment, the effects of archazolid have been intensively studied in cancer cells, but rarely in endothelial cells. Vioprolide is an antifungal and cytotoxic metabolite obtained from Cystobacter violaceus. There are only few studies on vioprolide, most of them focusing on its biosynthesis. Preliminary studies revealed that it inhibited TNF-induced expression of ICAM-1, indicating possible anti-inflammatory properties. As the endothelium plays an important role in cancer and inflammation, it represents an attractive drug target. Therefore, the archazolid and vioprolide were investigated regarding their effects on endothelial cells.
V-ATPase inhibition by archazolid resulted in anti-tumor and anti-metastatic effects in vitro and in vivo. Archazolid was used to study the consequences of v-ATPase inhibition in endothelial cells that might contribute to the anti-metastatic activities observed in vivo. To analyze the impact of archazolid on the interaction endothelial and cancer cells, in vitro cell adhesion and transmigration assays were performed using primary HUVEC or immortalized HMEC-1 and different cancer cell types (MDA-MB-231, PC-3 and Jurkat cells). For these experiments, only the endothelial cells were treated with archazolid. VATPase inhibition by archazolid led to an increased adhesion of the metastatic breast cancer cell line MDA-MB-231 and prostate cancer cell line PC-3 onto endothelial cells whereas the adhesion of Jurkat cells was unaffected. Interestingly, archazolid treatment of HUVECs decreased the transendothelial migration of MDA-MB-231 cells. Endothelial ICAM-1, VCAM-1, E-selectin and N-cadherin are potential ligands of interacting cancer cells. Therefore, the mRNA and surface protein levels of these cell adhesion molecules were measured via qRT-PCR and flow cytometry, respectively. These adhesion molecules were not responsible for the archazolid-induced cancer cell adhesion, as archazolid treatment of HUVECs did not upregulate their mRNA or surface expression. Instead, cell adhesion assays using a monoclonal antibody against integrin subunit β1 showed that β1-integrins expressed on MDA-MB-231 and PC-3 cells mediated the archazolid-induced cancer cell adhesion. Cell adhesion assays onto plastic coated with ECM components which are the major ligands of β1-integrins, revealed that MDA-MB231 and PC-3 cells preferably interact with collagen. So next, we investigated the influence of archazolid on surface collagen levels in HUVECs by immunostaining, which demonstrated an increase of nearly 50 % upon archazolid treatment. We confirmed the hypothesis that the expression and activity of cathepsin B, a lysosomal enzyme that degrades extracellular matrix components including collagen, was inhibited by archazolid in endothelial cells. Finally, overexpression of cathepsin B reduced the cancer cell adhesion on archazolid-treated HUVECs, but also in control cells, indicating a negative correlation between cathepsin B expression and cancer cell adhesion.
The influence of vioprolide on the interaction of endothelial cells with leukocytes was analyzed by in vitro cell adhesion assays using HUVECs and primary monocytes, THP-1 or Jurkat cells. Vioprolide inhibited the adhesion of these cells onto TNF-activated HUVECs. In addition, the endothelial-leukocyte interaction was observed in vivo by intravital microscopy in the mouse cremaster muscle. Vioprolide prevented the TNFinduced firm adhesion and transmigration of leukocytes, while leukocyte rolling was not affected. ICAM-1, VCAM-1 and E-selectin are cell adhesion molecules, which are upregulated by TNF and mediate leukocyte adhesion onto endothelial cells. Therefore, flow cytometric analysis was performed to measure their surface expression. Vioprolide significantly decreased TNF-induced expression of surface ICAM-1, VCAM-1 and E-selectin, which was in line with the in vitro results. In vivo, vioprolide may act in a different way on E-selectin expression, so that leukocyte rolling, which is governed by E-selectin, remained unaffected. qRT-PCR experiments revealed that the mRNA expression of ICAM-1 and VCAM-1 were also reduced by vioprolide, indicating a regulation on transcriptional level. In contrast, the mRNA expression of E-selectin was not decreased at the timepoint when surface protein expression was diminished. The induction of these cell adhesion molecules is mainly mediated by the transcription factor NFκB. A Dual-Luciferase® reporter assay was used to study the impact of vioprolide on the TNF-induced NFκB promotor activity. Vioprolide blocked the TNF-induced NFκB promotor activity while the TNF-induced IκBα degradation and nuclear translocation of the NFκB subunit p65 was not altered by vioprolide. Western blot analysis revealed that vioprolide had no effect on the activation of MAPK (p38, JNK) and AKT by TNF, which could interfere with the NFκB-dependent gene expression.
Taken together, archazolid and vioprolide are interesting myxobacterial compounds with different modes of actions. The study suggests that the v-ATPase inhibitor archazolid impairs the expression and activity of cathepsin B in endothelial cells, which leads to a higher amount of collagen on the endothelial surface. As a result, the adhesion of β1-integrin expressing metastatic cancer cells onto archazolid-treated endothelial cells increased while transendothelial migration was reduced. Further, archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Vioprolide was able to prevent TNF-induced endothelial-leukocyte interaction in vitro and in vivo by interfering with NFκB-dependent gene expression. Further research is required to enlighten the underlying mechanism and the direct target of vioprolide.
The overall survival for patients with acute lymphoblastic leukemia (ALL) often is the function of age, in particular in 2019 analysis revealed that 5-year overall survival for patients older than 20 years remains below 35% (American Cancer Society, Cancer Facts &Figures 2019). Importantly, one of the major issues in ALL therapy is the ability of tumor cells to escape the treatment via the establishment of an immunosuppressive environment. The tumor microenvironment has gained tremendous importance in the past decade. This is largely based on the reasoning that, in order to devise better therapeutic strategies for patients, we need to gain better understanding into how malignant cells transform their microenvironment to promote growth, escape immune control and gain therapeutic resistance.
TAM receptors (TAMRs) are engaged in innate immune cells as a feed-back mechanism to terminate the immune response and promote the return to homeostasis (Rothlin et al. 2007). In the context of cancers, aberrant TAMR signaling was mainly explored concerning its pro-oncogenic function (Paolino and Penninger 2016). There are only limited data available suggesting the modulation of cancer immune response via TAMR signaling in highly immunogenic solid tumor models (Paolino et al. 2014; Ubil et al. 2018). So far, however, little is known about their potential indirect immune-modulatory function in hematological malignancies. Taking into account the pronounced importance of TAMR signaling in immune cells combined with the leukemic immune tolerance, the current study focused on the function of TAMR and their ligands in anti-leukemic immunity.
This work uncovers the mechanism of dampening anti-leukemic immune response via TAMR signaling on macrophages using the syngeneic BCR-ABL1 B-ALL mouse model. Using genetic depletion of GAS6 in the host environment or ablation of AXL and/or MERTK receptors in macrophages the bone marrow microenvironment could be rewired in order to achieve an efficient anti-leukemic immune response. In particular, the GAS6/AXL blockade triggers an effective NKand T- cell-dependent anti-leukemic response that results in prolonged survival. This finding specifically tackles the obstacle of inefficient bridging between innate and adaptive immune response typical for hematological malignancies in contrast to solid tumors (E. K. Curran, Godfrey, and Kline 2017).
Besides establishing the vital function of TAMR signaling in anti-leukemic immunity using murine models, the analysis of human blood plasma revealed that age-related immune dysregulation was manifested by significant GAS6 decrease and PROS1 upregulation among elderly donors (>60 y.o.) compared to controls (<25 y.o.). These data are indicative that TAMR signaling likely favors the age-dependent immune system decline, which in turn is associated with a poor survival rate of elderly patients diagnosed with leukemia.
In conclusion, using a preclinical ALL model here it was identified in vivo, that Axl significantly increases upon B-ALL challenge in Mph and NK cells. Therefore, AXL targeting, using the orally bioavailable selective inhibitor Bemcentinib, could serve as a powerful approach to revert early immunosuppression created by leukemia.
Taken together these data propose the AXL receptor as a novel immune checkpoint and attractive candidate for the development of a new therapeutic approach via unleashing the patient’s own immune system to combat leukemic cells.
T-cell development is a highly dynamic and stepwise process comprimising T lineage commitment, T-cell receptor (TCR) gene rearrangements and subsequent selection. From a quantitative point of view, only a few hundred progenitor cells migrate from the bone marrow into the thymus. Developing thymocytes (termed double negative (DN), CD4-CD8-) can be further divided into DN1-4 cells based on the expression of CD25 and CD44. These developmental events are interspersed by proliferative bursts which ultimately lead to the generation of millions of double positive (DP, CD4+CD8+) thymocytes that then undergo selection. As a consequence, a proportion of naïve T-cells evolves to ensure adaptive, but not autoreactive immunity.
Previous studies of our lab focused on the quantification of thymus colonization and identified thymus entry to be dependent on expression of the chemokine receptors CCR7 and CCR9 (Krueger et al., 2010; Ziętara et al., 2015). CCR7/9 double knockout (DKO) mice are almost completely devoid of the most immature thymocyte populations (DN1 and DN2), but show near normal DN3 cellularity. Interestingly, a similar defect during early development but a virtually complete recovery of later stages and total thymocyte numbers was also observed in thymi of miR-17~92 deficient mice. Here, a failure of prethymic IL-7 signaling dampens early T-cell development (Regelin et al., 2015). For this reason, we hypothesized a tight regulation of thymocyte population size through alterations in the underlying cell cycle kinetics.
In this thesis, we employed in vivo single- and dual-nucleoside pulse labeling combined with determination of DNA replication over time in different WT thymocyte subsets at steady-state. Based on this, we assessed alterations in cell cycle kinetics of CCR7/9 and miR-17~92 defcicient mice and identified compensatory mechanisms of thymocytes on the level of cell cycle phase distribution and cell cycle speed. In addition, single-cell RNA sequencing helped to obtain information on cell cycle dynamics of early thymocyte subsets, exemplarily shown for WT and CCR7/9 DKO mice. Lastly, we performed cell cycle analyses in a model of endogenous thymic repair upon sublethal total body irradiation which provided insight into intrathymic cell cycle regulation as an adjustable system to re-establish normal thymus cellularity.
In the second part of the thesis, we addressed the role of miR-21 in the thymus. In various studies, we and others identified miRNAs as key posttranscriptional regulators of the immune system and especially for T-cell development (Regelin et al. 2015; Mildner et al. 2017; Li et al. 2007; Ebert et al. 2009; Ziętara et al. 2013; Schaffert et al. 2015). The dynamic expression of miR-21 during T-cell development (Neilson et al. 2007; Kirigin et al. 2012; Kuchen et al. 2010) prompted us to hypothesize that miR-21 has a regulatory function in the thymus. A miR 21-knockout mouse model allowed us to study the role of this miRNA for the development of T-cells in the thymus and the maintenance of T-cells in the periphery. In addition, we performed competitive bone marrow chimera experiments in the context of miR-21 deficiency and overexpression. Further insights were provided by exploring the function of miR-21 in negative selection in vivo as well as in T-cell differentiation in coculture experiments in vitro. To unravel implications of miR-21 to regulate cellular stress responses, we assessed the contribution of miR-21 in a model of endogenous regeneration of the thymus after sublethal irradiation. We could not provide evidence for a prominent role for miR-21 during T-cell development. Together, our experiments revealed that miR-21 is largely dispensable for physiologic T-cell development despite high and dynamic expression in the thymus (Kunze Schumacher et al., 2018). The apparent discrepancy between dynamic expression but lack of a regulatory function in the thymus led us to conclude that miR-21 is rather fine tuning T-cell responses than controlling a developmental event.
Identifizierung potenzieller Taspase1 Inhibitoren für die Behandlung von t(4,11) akuter Leukämie
(2022)
Leukämie ist die häufigste bösartige Krebserkrankung im Kindes- und Jugendalter. Bei einem Kind von 1120 Kindern wird Leukämie diagnostiziert, dabei trifft diese Diagnose Jungen 30 % häufiger als Mädchen. Die Krankheitssymptome treten bei den Kindern noch vor dem Schulalter auf und am häufigsten haben die Kinder mit der akuten Form zu kämpfen. Bei einer Diagnose mit einer akuten lymphatischen Leukämie (ALL) haben die Kinder meist eine gute Prognose, während bei der akuten myeloischen Leukämie (AML) eine deutlich schlechtere.1
Die t(4;11)(q21;q23) Translokation ist aufgrund ihres häufigen Auftretens und der damit schlechten verbundenen Prognose eines der bekanntesten strukturellen Chromosomen-anomalien bei akuten Leukämien. Diese Translokation wurde das erste Mal 1977 von Oshimura et al. beschrieben.2 Bei einer t(4;11)-Translokation ist das Chromosom 4 und das Chromosom 11 involviert. Auf Chromosom 4 ist das AF4-Gen lokalisiert (AFF1) und auf dem Chromosom 11 liegt das MLL-Gen (ALL-1, HRX, hTRX, KMT2A).
Taspase1 wurde als ein proteolytisch prozessierendes Enzym identifiziert, das sich in Wirbellosen und Vertebraten zusammen mit Mitgliedern der Trithorax/MLL/KMT2A-Protein¬familie koevolviert hat. Taspase1 prozessiert nicht nur das MLL und MLL2, deren Fusions¬proteine AF4-MLL, sondern auch den Transkriptionsfaktor IIA (TFIIA) sowohl in vitro als auch in vivo.3
Die Dimerisierung von Taspase1 löst eine intrinsische Serinproteasefunktion aus, die zum katalytischen Rest Thr234 führt, der die Konsensussequenz Q-3X-2D-1•G1X2D3D4 katalysiert, die in Mitgliedern der MLL-Familie sowie im Transkriptionsfaktor TFIIA vorhanden ist. Taspase1 ist kein klassisches Enzym, da es seine Zielproteine stöchiometrisch hydrolysiert. Diese Eigenschaft macht es nahezu unmöglich, in einem klassischen Screening-Setup nach potenziellen Inhibitoren zu screenen.
In dieser Arbeit wurde ein Homogeneous time-resolved fluorescence HTRF-Reporter-Assays etabliert. Das etablierte Testsystem ermöglicht erstmalig die Untersuchung von Substanzen zusammen mit Taspase1 Monomere, die in einem zellfreien System (cfs) hergestellt wurden. Durch die Expression non monomeren Taspase1 Proteinen sollten Inhibitoren durch das etablierte Screening-Verfahren gefunden werden, die sowohl (1) Dimerisierung, (2) Autoaktivierung oder (3) Substratbindung selektiv blockieren können. Die durchgeführten Experimente führten zur Identifikation eines ersten Taspase1-Inhibitors, Closantel sodium. Closantel sodium ist ein U.S. Food and Drug Administration (FDA) zugelassenes Medikament, das Taspase1 auf nicht-kovalente Weise bindet. Die erzielten Daten zeigen, dass Closantel sodium den Dimerisierungsschritt und/oder die intrinsische Serinproteasefunktion blockiert. Closantel sodium hemmte die Spaltung des eingesetzten CS2-Substratproteins mit einem IC50 zwischen 1,6 und 3,9 µM, je nachdem, welches Taspase1-Präparat in dem HTRF Screening Assay ver¬wendeten (cfs- oder E.coli-produziert). Die Daten weisen darauf hin, dass Closantel sodium als allosterischer Inhibitor gegen die Taspase1 fungiert. Taspase1 wird zur Aktivierung der AF4-MLL-Onkofusionsproteine benötigt und wird auch in mehreren soliden Tumoren überexprimiert. Daher könnte dieser neue Inhibitor für die weitere Validierung von Taspase1 als Ziel für die Krebstherapie und für das Design potenterer Liganden für zukünftige klinische Anwendungen nützlich sein.
The specific and precise arrangement of proteins and biomolecules in 3D is an important prerequisite for the study of cell migration, cellular signal transduction and the production of artificial tissue. In a variety of research approaches, proteins have been immobilized on rigid surfaces such as glass or gold to observe protein-protein or protein-cell interactions. While these commonly used analytical platforms offer advantages such as rapid washing steps and easy use, due to their rigidity and two-dimensionality, they cannot replicate the extracellular matrix (ECM) the native environment of cells. This severe deviation from the natural environment results in significant changes in cell structure and cellular processes such as the polarization of the cell, its morphology, and signal transduction. In order to maintain the functionality of the immobilized proteins, it is also enormously important that the proteins are oriented and anchored in the material under mild conditions.
An immobilization strategy that makes this possible is bioaffinity. For this, the specific interaction of a biomolecule with an interaction partner anchored on a surface is used to immobilize the biomolecule. Such an interaction is for example the nitrilotriacetic acid (NTA)/His-tag binding. NTA is a chelator molecule that, when bound to divalent metal ions such as Ni(II), forms an octahedral complex with oligohistidines. The oligo histidine-tag can be competed out of the complex by free histidine or imidazole due to structural similarity. This is exploited in immobilized metal affinity chromatography (IMAC). The binding of a monoNTA/His-tag complex (KD=10 µM) is not stable enough to be used for immobilizations. Therefore, multivalent variants of the chelator were developed, like trisNTA which has a high affinity for His6 tagged proteins (KD= 10 nM). The PA-trisNTA developed in a preliminary work was the first light-activatable system based on the trisNTA chelator head.
The aim of this work was to synthesize a new two-photon (2P) activatable trisNTA (TPA trisNTA) interaction molecule, to analyze its photophysical characteristics and to apply it for two- and three dimensional (2D/3D) biomolecule patterning. The final goal was to use TPA trisNTA for cellular applications in order to manipulate membrane protein organization. Therefore, TPA trisNTA was designed to maintain a stable autoinhibition enabling the immobilization of proteins under physiological conditions with high precision in the x/y, as well as z dimension only upon light activation. 2P activation brings some outstanding advantages: i) the use of near-infrared (NIR) light is less harmful to cells compared to ultraviolet (UV) light, ii) the longer wavelength allows the radiation to penetrate deeper into tissues, iii) the precision of focal irradiation is more accurate because only a focal volume (about 1 fL) is excited and, unlike UV light, scattered light does not lead to activation.
Several backbones for TPA-trisNTA were considered as 2P cleavable groups due to their 2P absorption ability and small size: 3 nitrodibenzofuran (NDBF), 6 bromo 7 hydroxycoumarin (Bhc), and 7 diethylaminocoumarin (DEAC). Initially, suitable synthetic routes were developed for the respective carbaldehydes, since these represented an important intermediate for both the construction of amino acid (aa) derivatives as well as ß hydroxy acids. ß Hydroxy acids were important intermediates because their photocleavage differs from aa derivatives. To establish the conversion from carbaldehydes to hydroxy acids via Reformatsky reaction, commercially available carbaldehydes of the nitroveratral (NV) or nitropiperonal (NP) group were used in addition. The conversion of NDBF, NV, NP proved to be difficult, whereas the ß-hydroxy acid was successfully synthesized from Bhc as well as from DEAC.
Starting from DEAC ß hydroxy acid, a Fmoc protected amino acid derivative was synthesized. To ensure high cleavage efficiency, the DEAC ß hydroxy acid was linked to monoFmoc ethylenediamine through a carbamate linker. Subsequently, the photocleavable group was successfully incorporated into the linker of TPA-trisNTA by solid-phase peptide synthesis (SPPS).
The functional principle of TPA-trisNTA, similar to PA-trisNTA, is based on the autoinhibition of the multivalent chelator head trisNTA, which is linked to an intramolecular oligohistidine sequence by a peptide linker. In presence of Ni(II) ions, trisNTA forms a metal ion-mediated complex with histidine, causing TPA-trisNTA to self-inactivate. The cleavage site is the DEAC based photocleavable amino acid. In contrast to PA-trisNTA, the incorporation of two photocleavable amino acids was omitted. Instead, only one photocleavable DEAC was incorporated in front of the His tag. To avoid a second DEAC group within the His tag, a His5 tag was used instead of an His6 tag. It is known from preliminary work that a His5 tag is sufficient to maintain autoinhibition in the presence of His6-tagged proteins of interest (POIs), but can be displaced from the complex after light-driven cleavage of the peptide backbone. Placement of a cysteine in the peptide linker between the trisNTA and the DEAC group allowed for permanent surface anchoring after photocleavage of the linker.
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Treatment of hexachloropropene (Cl2C[double bond, length as m-dash]C(Cl)–CCl3) with Si2Cl6 and [nBu4N]Cl (1 : 4 : 1) in CH2Cl2 results in a quantitative conversion to the trisilylated, dichlorinated allyl anion salt [nBu4N][Cl2C[double bond, length as m-dash]C(SiCl3)–C(SiCl3)2] ([nBu4N][1]). Tetrachloroallene Cl2C[double bond, length as m-dash]C[double bond, length as m-dash]CCl2 was identified as the first intermediate of the reaction cascade. In the solid state, [1]− adopts approximate Cs symmetry with a dihedral angle between the planes running through the olefinic and carbanionic fragments of [1]− of C[double bond, length as m-dash]C–Si//Si–C–Si = 78.3(1)°. One-electron oxidation of [nBu4N][1] with SbCl5 furnishes the distillable blue radical 1˙. The neutral propene Cl2C[double bond, length as m-dash]C(SiCl3)–C(SiCl3)2H (2) was obtained by (i) protonation of [1]− with HOSO2CF3 (HOTf) or (ii) H-atom transfer to 1˙ from 1,4-cyclohexadiene. Quantitative transformation of all three SiCl3 substituents in 2 to Si(OMe)3 (2OMe) or SiMe3 (2Me) substituents was achieved by using MeOH/NMe2Et or MeMgBr in CH2Cl2 or THF, respectively. Upon addition of 2 equiv. of tBuLi, 2Me underwent deprotonation with subsequent LiCl elimination, 1,2-SiMe3 migration and Cl/Li exchange to afford the allenyl lithium compound Me3Si(Li)C[double bond, length as m-dash]C[double bond, length as m-dash]C(SiMe3)2 (Li[4]), which is an efficient building block for the introduction of Me, SiMe3, or SnMe3 (5) groups. The trisilylated, monochlorinated allene Cl3Si(Cl)C[double bond, length as m-dash]C[double bond, length as m-dash]C(SiCl3)2 (6), was obtained from [nBu4N][1] through Cl−-ion abstraction with AlCl3 and rearrangement in CH2Cl2 (1˙ forms as a minor side product, likely because the system AlCl3/CH2Cl2 can also act as a one-electron oxidant).
Photoacids attract increasing scientific attention, as they are valuable tools to spatiotemporally control proton-release reactions and pH values of solutions. We present the first time-resolved spectroscopic study of the excited state and proton-release dynamics of prominent merocyanine representatives. Femtosecond transient absorption measurements of a pyridine merocyanine with two distinct protonation sites revealed dissimilar proton-release mechanisms: one site acts as a photoacid generator as its pKa value is modulated in the ground state after photoisomerization, while the other functions as an excited state photoacid which releases its proton within 1.1 ps. With a pKa drop of 8.7 units to −5.5 upon excitation, the latter phenolic site is regarded a super-photoacid. The 6-nitro derivative exhibits only a phenolic site with similar, yet slightly less photoacidic characteristics and both compounds transfer their proton to methanol and ethanol. In contrast, for the related 6,8-dinitro compound an intramolecular proton transfer to the ortho-nitro group is suggested that is involved in a rapid relaxation into the ground state.
The knob-associated histidine-rich protein (KAHRP) plays a pivotal role in the pathophysiology of Plasmodium falciparum malaria by forming membrane protrusions in infected erythrocytes, which anchor parasite-encoded adhesins to the membrane skeleton. The resulting sequestration of parasitized erythrocytes in the microvasculature leads to severe disease. Despite KAHRP being an important virulence factor, its physical location within the membrane skeleton is still debated, as is its function in knob formation. Here, we show by super-resolution microscopy that KAHRP initially associates with various skeletal components, including ankyrin bridges, but eventually colocalizes with remnant actin junctions. We further present a 35 Å map of the spiral scaffold underlying knobs and show that a KAHRP-targeting nanoprobe binds close to the spiral scaffold. Single-molecule localization microscopy detected ~60 KAHRP molecules/knob. We propose a dynamic model of KAHRP organization and a function of KAHRP in attaching other factors to the spiral scaffold.
Komplexe biologische Phänotypen resultieren aus einem koordinierten Zusammenspiel von einer Vielzahl von Genen. Um zu verstehen, wie Krankheiten durch genetische Dysfunktionen
entstehen können, ist es unabdingbar die genetischen Interaktionsnetzwerke in menschlichen Zellen zu entschlüsseln. Eine Identifizierung von Kontext-abhängigen genetischen Interaktionen kann bedeutende Erkenntnisse über die Beziehung von Phänotyp und Genotyp liefern und erklären, wie synergistische Gen-Funktionen die Entstehung von komplexen Krankheiten bedingen.
Gepoolte, kombinatorische CRISPR (kurz für: clustered regularly interspaced short palindromic repeats) Screens stellen eine wirkungsvolle Methode zur simultanen Untersuchung potentieller Interaktionen von einer großen Anzahl von Genen dar. Mit sogenannten multiplex CRISPR
gRNA Bibliotheken werden im Rahmen großangelegter Screens vielzählige kombinatorische Gen-Knockouts in Zellen generiert. Diese multiplex CRISPR gRNA Bibliotheken können aus bis zu hunderttausenden Plasmiden bestehen, die jeweils für eine andere gRNA-Kombination kodieren und auf ein spezifisches Gen-Paar abzielen. Im Gegensatz zu CRISPR Screens für Einzel-Knockouts gehen multiplex CRISPR Screens zur Identifizierung von genetischen Interaktionen mit zusätzlichen Herausforderungen einher: Zum einen wächst der verbundene Arbeitsaufwand für die Konstruktion der multiplex CRISPR gRNA Bibliotheken proportional mit der Anzahl der gewünschten Ziel-Gene, welche die Diversität der Bibliothek bestimmt. In einer idealen gRNA-Bibliothek wären alle gRNA-Sequenzen gleich häufig vorhanden. Jedoch weisen
gRNA-Bibliotheken aufgrund von technischen Beschränkungen gRNA-Sequenzen mit höherer, beziehungsweise niedriger Abundanz auf. Konventionelle Methoden zur Herstellung von
gRNA-Bibliotheken basieren beispielsweise auf iterativen, gepoolten Klonierungsschritten mit PCR-amplifizierten Oligonucleotiden, welche zu einer Ungleichverteilung oder zum Verlust von gRNA-Sequenzen führen können. Daher bieten Methoden zur gRNA-Bibliotheken-Generierung Optimierungspotenzial. Da die Reproduzierbarkeit der Screen-Ergebnisse durch die sogenannte Screening Coverage sichergestellt werden muss, erfordert eine Erhöhung der
Bibliotheks-Diversität gleichzeitig auch eine Vergrößerung des Versuchsmaßstabs und ist mit umfangreichem Zellkultur-Arbeitsaufwand verbunden. Die Screening Coverage gibt die
durchschnittliche Abundanz der einzelnen gRNA-Sequenzen in der Zellpopulation während des Screens an. Aktuelle Richtlinien empfehlen eine Screening Coverage, die zwischen dem 200- bis 1000-fachen Wert der Bibliotheks-Diversität liegt, allerdings fehlen bisher genaue Angaben die auf die verwendete gRNA Bibliothek abgestimmt sind. Deshalb stellt die benötigte Screening Coverage bisher einen limitierenden Faktor dar, der die Anzahl der möglichen Ziel-Gene-Kombinationen in einem Screen beschränkt.
In der vorliegenden Arbeit stellen wir eine neue Methode zur Generierung von multiplex gRNA Bibliotheken mit hohen Diversitäten vor. Die Methode, genannt 3Cs (covalently-closed circular-synthesized) Multiplexing, umgeht iterative, gepoolte Klonierugsschritte mit Restriktionsenzymen und PCR-Amplifikation von gRNA-kodierenden Oligonucleotiden. Wir
zeigen, dass 3Cs Multiplexing auf robuste Weise zur Herstellung von gleichmäßig verteilten multiplex gRNA Bibliotheken verwendet werden kann. Der Verteilungs-Skew, auch Skew-Ratio oder Bibliotheksbreite genannt, ist ein Maß zur Ermittlung der Gleichverteilung der gRNA-Sequenzen in der Bibliothek. Wir zeigen, dass 3Cs multiplex Bibliotheken typischerweise einen Verteilungs-Skew von 2.5 aufweisen, was unter den üblichen Werten von Einzel-gRNA Bibliotheken liegt.
Wir nahmen an, dass die gRNA-Bibliotheksverteilung die Robustheit von gepoolten CRISPR Screens beeinflussen könne und deshalb bei der Auswahl einer geeigneten Screening
Coverage berücksichtigt werden müsse. Um den Einfluss der gRNA-Bibliotheksverteilung auf die Screen-Qualität in Abhängigkeit von der verwendeten Screening Coverage zu untersuchen, generierten wir zwei künstlich fehlverteilte multiplex gRNA-Bibliotheken. Diese wurden, zusätzlich zu einer nahezu gleichverteilten multiplex gRNA-Bibliothek, jeweils mit einer 20- und 200-fachen Screening Coverage in einem kombinatorischen Proliferationsscreen angewandt.
Dadurch konnten wir die gRNA-Bibliotheksverteilung als den bestimmenden Parameter für die benötigte Screening Coverage identifizieren. Zusätzlich konnten wir zeigen, dass 3Cs multiplex gRNA-Bibliotheken auf Grund ihrer gleichmäßigen Verteilung mit minimierter Screening Coverage eingesetzt werden können, was zu einer 10-fachen Reduktion des assoziierten Arbeitsaufwands führt. Während bisherige Richtlinien für gepoolte CRISPR Screens die initiale
gRNA-Bibliotheksverteilung nicht berücksichtigen, empfehlen wir die Screening Coverage an dieser auszurichten.
Autophagie ist ein streng regulierter zellulärer Prozess, der den Lysosomen Abbau von intrazellulärem Material steuert und im Zusammenhang mit zahlreichen menschlichen Erkrankungen steht. Da Autophagie in eine Vielzahl von Signalwegen integriert ist, bietet es außerdem therapeutische Ansatzpunkte zur Behandlung von Krankheiten. Die Identifizierung von synergistischen Funktionen zwischen Autophagie-Genen könnte unser Verständnis über die molekularen Mechanismen, die der Regulation der Autophagie zu Grunde liegen, erweitern und dadurch neuartige Behandlungen ermöglichen.
Um genetische Interaktionen von Autophagie-Genen zu untersuchen haben wir eine 3Cs multiplex gRNA Bibliothek generiert, die auf menschliche Autophagie-Genkombinationen
abzielt. In dieser Arbeit demonstrieren wir die Funktionalität der 3Cs Autophagie multiplex gRNA Bibliothek unter Anwendung minimierter Screening Coverage in zwei verschiedenen Screen-Ausführungen: In einem Proliferationsscreen konnten wir Geninteraktionen
identifizieren, deren Verlust zu einer gesteigerten oder verringerten Zellproliferation führt. Unter diesen resultierte der Knockout von WDR45B-PIK3R4 zur stärksten Suppression der Proliferation, während die Depletion von ATG7-KEAP1 zu extrem verstärkter Proliferation beitrug. Unter Einsatz eines Autophagie-Reporters konnten wir in einem Autophagie Screen genetische Interaktionen aufdecken, die essentiell für Autophagie sind, darunter die
Interaktionen zwischen ATG2A-ATG2B , GABARAPL2-WIPI2 und ULK4-SQSTM1.
Wir glauben, dass 3Cs Multiplexing in Zukunft breite Anwendung in verschiedenen biologisch relevanten Feldern finden kann und die Entschlüsselung von kontext-abhängigen genetischen Interaktionen voranbringen und so das Verständnis für die Entstehung von komplexen pathologischen Phänotypen erweitern wird.
In vivo inducible reverse genetics in patients' tumors to identify individual therapeutic targets
(2021)
High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.
A highly diastereoselective one-pot synthesis of the 1,3-diamino-2-alcohol unit bearing three continuous stereocenters is described. This method utilizes 2-oxyenamides as a novel type of building block for the rapid assembly of the 1,3-diamine scaffold containing an additional stereogenic oxygen functionality at the C2 position. A stereoselective preparation of the required (Z)-oxyenamides is reported as well.
Designed polypharmacology presents as an attractive strategy to increase therapeutic efficacy in multi-factorial diseases by a directed modulation of multiple involved targets with a single molecule. Such an approach appears particularly suitable in non-alcoholic steatohepatitis (NASH) which involves hepatic steatosis, inflammation and fibrosis as pathological hallmarks. Among various potential pharmacodynamic mechanisms, activation of the farnesoid X receptor (FXRa) and inhibition of leukotriene A4 hydrolase (LTA4Hi) hold promise to counteract NASH according to preclinical and clinical observations. We have developed dual FXR/LTA4H modulators as pharmacological tools, enabling evaluation of this polypharmacology concept to treat NASH and related pathologies. The optimized FXRa/LTA4Hi exhibits well-balanced dual activity on the intended targets with sub-micromolar potency and is highly selective over related nuclear receptors and enzymes rendering it suitable as tool to probe synergies of dual FXR/LTA4H targeting.
The desensitized channelrhodopsin-2 photointermediate contains 13 -cis, 15 -syn retinal Schiff base
(2021)
Channelrhodopsin-2 (ChR2) is a light-gated cation channel and was used to lay the foundations of optogenetics. Its dark state X-ray structure has been determined in 2017 for the wild-type, which is the prototype for all other ChR variants. However, the mechanistic understanding of the channel function is still incomplete in terms of structural changes after photon absorption by the retinal chromophore and in the framework of functional models. Hence, detailed information needs to be collected on the dark state as well as on the different photointermediates. For ChR2 detailed knowledge on the chromophore configuration in the different states is still missing and a consensus has not been achieved. Using DNP-enhanced solid-state MAS NMR spectroscopy on proteoliposome samples, we unambiguously determined the chromophore configuration in the desensitized state, and we show that this state occurs towards the end of the photocycle.
Polymorphic G-quadruplex (G4) secondary DNA structures have received increasing attention in medicinal chemistry owing to their key involvement in the regulation of the maintenance of genomic stability, telomere length homeostasis and transcription of important proto-oncogenes. Different classes of G4 ligands have been developed for the potential treatment of several human diseases. Among them, the carbazole scaffold with appropriate side chain appendages has attracted much interest for designing G4 ligands. Because of its large and rigid π-conjugation system and ease of functionalization at three different positions, a variety of carbazole derivatives have been synthesized from various natural or synthetic sources for potential applications in G4-based therapeutics and biosensors. Herein, we provide an updated close-up of the literatures on carbazole-based G4 ligands with particular focus given on their detailed binding insights studied by NMR spectroscopy. The structure-activity relationships and the opportunities and challenges of their potential applications as biosensors and therapeutics are also discussed. This review will provide an overall picture of carbazole ligands with remarkable G4 topological preference, fluorescence properties and significant bioactivity; portraying carbazole as a very promising scaffold for assembling G4 ligands with a range of novel functional applications.
Chemical language models enable de novo drug design without the requirement for explicit molecular construction rules. While such models have been applied to generate novel compounds with desired bioactivity, the actual prioritization and selection of the most promising computational designs remains challenging. Herein, we leveraged the probabilities learnt by chemical language models with the beam search algorithm as a model-intrinsic technique for automated molecule design and scoring. Prospective application of this method yielded novel inverse agonists of retinoic acid receptor-related orphan receptors (RORs). Each design was synthesizable in three reaction steps and presented low-micromolar to nanomolar potency towards RORγ. This model-intrinsic sampling technique eliminates the strict need for external compound scoring functions, thereby further extending the applicability of generative artificial intelligence to data-driven drug discovery.
Two subvalent, redox-active diborane(4) anions, [3]4− and [3]2−, carrying exceptionally high negative charge densities are reported: Reduction of 9-methoxy-9-borafluorene with Li granules without stirring leads to the crystallization of the B(sp3)−B(sp2) diborane(5) anion salt Li[5]. [5]− contains a 2,2′-biphenyldiyl-bridged B−B core, a chelating 2,2′-biphenyldiyl moiety, and a MeO substituent. Reduction of Li[5] with Na metal gives the Na+ salt of the tetraanion [3]4− in which two doubly reduced 9-borafluorenyl fragments are linked via a B−B single bond. Comproportionation of Li[5] and Na4[3] quantitatively furnishes the diborane(4) dianion salt Na2[3], the doubly boron-doped congener of 9,9′-bis(fluorenylidene). Under acid catalysis, Na2[3] undergoes a formal Stone–Wales rearrangement to yield a dibenzo[g,p]chrysene derivative with B=B core. Na2[3] shows boron-centered nucleophilicity toward n-butyl chloride. Na4[3] produces bright blue chemiluminescence when exposed to air.
SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.
Diversity-oriented synthesis (DOS) is a rich source for novel lead structures in Medicinal Chemistry. In this study, we present a DOS-compatible method for synthesis of compounds bearing a free thiol moiety. The procedure relies on Rh(II)-catalyzed coupling of dithiols to diazo building blocks. The synthetized library was probed against metallo-β-lactamases (MBLs) NDM-1 and VIM-1. Biochemical and biological evaluation led to identification of novel potent MBL inhibitors with antibiotic adjuvant activity.
B-cell acute lymphoblastic leukaemia (B-ALL) is characterized by the overproduction of lymphoblasts in the bone marrow (BM), and it is the most common cancer in children while being comparatively uncommon in adults. On the other hand, in chronic myeloid leukaemia (CML), 70% of cases are found in patients older than 50 years, making it uncommon in children. All CML cases and up to 3% of paediatric B- ALL (and 25% of adult B-ALL) cases are due to fusion gene BCR-ABL1, which gives rise to the cytoplasmatic, constitutively active oncoprotein, tyrosine kinase BCR-ABL1 through a reciprocal translocation between chromosomes 9 and 22. The constitutively active BCR-ABL tyrosine kinase leads to deregulation of different signal transduction pathways such as cell growth, proliferation and cell survival. The role of the bone marrow microenvironment (BMM) can mediate disease initiation (only in mice), progression, therapy resistance, and relapse, as has been increasingly recognized over the last two decades. In general, the BMM is a very complex arrangement of various cell types such as osteoblasts, osteoclasts, endothelial cells, adipocytes, mesenchymal stromal cells, macrophages and several others. In addition, the BMM is composed of multiple chemical and mechanical factors and extra cellular matrix (ECM) proteins which contribute to the BMM’s features influencing leukaemia behaviour. Considering the incidence of B-ALL and CML in children and in adults respectively, we hypothesized that the young and/or an aged BMM might also play a previously unrecognized role in the aggressiveness of B-ALL and CML. We proposed that BM, transduced with BCR-ABL1-expressing retrovirus in the murine transduction/transplantation model of B-ALL, transplanted into young versus old recipient mice would lead to a more aggressive disease in young mice, and similarly CML would be more aggressive in old recipient mice. In close recapitulation with the human incidence, induction of CML led to a significantly shorted survival in old recipient mice. On the other hand, induction of B-ALL showed a shortened survival in young compared to old syngeneic mice, as well as in a xenotransplantation model. Among the highly heterogenous composition of the BMM, we implicate young BM macrophages as a supportive niche for B-ALL cells. The results were found to be mostly due to potential soluble factors differentially secreted from young and old macrophages. Therefore, we hypothesized that the chemokine CXCL13, which has been demonstrated to play a role in B cell migration and act as a diagnostic marker in the cerebrospinal fluid of patients with neuroborreliosis, might be responsible for the observed phenotype. CXCL13 was found to be more highly expressed in healthy and leukaemic young mice as well as in conditioned medium of young macrophages. Using a variety of in vitro experiments, CXCL13 showed to significantly increase the proliferation and the migration of leukaemia cells when exposed to young macrophages, and the phenotype was rescued while using a CXCL13 neutralizing antibody. The CXCL13 role was also confirmed in vivo, since macrophage ablation led to a prolongation of survival in young mice and a reduction of CXCL13 levels. The use of an additional mouse model, leukaemia cells with CXCR5 deficiency, led to a significant prolongation of survival of young mice, confirming the importance of the CXCL13-CXCR5 axis in B-ALL. In line with our murine results, we found that human macrophages and CXCL13 levels were higher in pediatric B-ALL patients than in adults. Consistent with our murine data, the expression level of CXCR5 may act as a prognostic marker in B-ALL, as well as a predictive marker for central nervous system relapse in human B-ALL. The overall findings show that a young BMM, and in particular macrophages, influences B-ALL progression. We specifically identified CXCL13, secreted by young macrophages, as a promoter of proliferation of B-ALL cells, influencing survival in B-ALL via CXCR5. The CXCR5-CXCL13 axis may be relevant in human B-ALL, and higher CXCR5 expression in human B-ALL may act as a predictive marker.
Precise control of blood clotting and rapid reversal of anticoagulation are essential in many clinical situations. We were successful in modifying a thrombin-binding aptamer with a red-light photocleavable linker derived from Cy7 by Cu-catalyzed Click chemistry. We were able to show that we can successfully deactivate the modified aptamer with red light (660 nm) even in human blood—restoring the blood's natural coagulation capability.
In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples.
The intriguing (μ-hydrido)diboranes(4) with their prominent pristine representative [B2H5]− have mainly been studied theoretically. We now describe the behavior of the planarized tetraaryl (μ-hydrido)diborane(4) anion [1H]− in cycloaddition reactions with the homologous series of heterocumulenes CO2, iPrNCO, and iPrNCNiPr. We show that a C=O bond of CO2 selectively activates the B−B bond of [1H]−, while the μ-H ligand is left untouched ([2H]−). The carbodiimide iPrNCNiPr, in contrast, neglects the B−B bond and rather adds the B-bonded H− ion to its central C atom to generate a formamidinate bridge across the B2 pair ([3]−). As a hybrid, the isocyanate iPrNCO combines the reactivity patterns of both its congeners and gives two products: one of them ([4H]−) is related to [2H]−, the other ([5]−) is an analog of [3]−. We finally propose a mechanistic scenario that rationalizes the individual reaction outcomes and combines them to a coherent picture of B–B vs. B–H bond activation.
The crystal structures of sodium ethoxide (sodium ethanolate, NaOEt), sodium n-propoxide (sodium n-propanolate, NaOnPr), sodium n-butoxide (sodium n-butanolate, NaOnBu) and sodium n-pentoxide (sodium n-amylate, NaOnAm) were determined from powder X-ray diffraction data. NaOEt crystallizes in space group P421m, with Z = 2, and the other alkoxides crystallize in P4/nmm, with Z = 2. To resolve space-group ambiguities, a Bärnighausen tree was set up, and Rietveld refinements were performed with different models. In all structures, the Na and O atoms form a quadratic net, with the alkyl groups pointing outwards on both sides (anti-PbO type). The alkyl groups are disordered. The disorder becomes even more pronounced with increasing chain length. Recrystallization from the corresponding alcohols yielded four sodium alkoxide solvates: sodium ethoxide ethanol disolvate (NaOEt·2EtOH), sodium n-propoxide n-propanol disolvate (NaOnPr·2nPrOH), sodium isopropoxide isopropanol pentasolvate (NaOiPr·5iPrOH) and sodium tert-amylate tert-amyl alcohol monosolvate (NaOtAm·tAmOH, tAm = 2-methyl-2-butyl). Their crystal structures were determined by single-crystal X-ray diffraction. All these solvates form chain structures consisting of Na+, –O− and –OH groups, encased by alkyl groups. The hydrogen-bond networks diverge widely among the solvate structures. The hydrogen-bond topology of the iPrOH network in NaOiPr·5iPrOH shows branched hydrogen bonds and differs considerably from the networks in pure crystalline iPrOH.
Cytochrome c oxidases are among the most important and fundamental enzymes of life. Integrated into membranes they use four electrons from cytochrome c molecules to reduce molecular oxygen (dioxygen) to water. Their catalytic cycle has been considered to start with the oxidized form. Subsequent electron transfers lead to the E-state, the R-state (which binds oxygen), the P-state (with an already split dioxygen bond), the F-state and the O-state again. Here, we determined structures of up to 1.9 Å resolution of these intermediates by single particle cryo-EM. Our results suggest that in the O-state the active site contains a peroxide dianion and in the P-state possibly an intact dioxygen molecule, the F-state may contain a superoxide anion.
Cerumen was found to be a promising alternative specimen for the detection of drugs. In a pilot study, drugs of abuse were identified at a higher detection rate and a longer detection window in cerumen than in urine. In this study, cerumen from subjects was analyzed after they ingested the designer stimulant 4-fluoroamphetamine (4-FA) in a controlled manner. Methods: Twelve subjects ingested placebo and 100 mg of 4-FA. Five of them were also given 150 mg of 4-FA in 150 mL Royal Club bitter lemon drink at least after 7 days. Cerumen was sampled using cotton swabs at baseline, 1 h after the ingestion of the drug and at the end of the study day (12 h). After extraction with ethyl acetate followed by solid-phase extraction, the extracts were analyzed using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Results and discussion: In the cerumen of all 12 subjects, 4-FA was detected 12 h after its ingestion; in most subjects, cerumen was detected after 1 h of ingestion, ranging from 0.06 to 13.90 (median 1.52) ng per swab. The detection of 4-FA in cerumen sampled 7 days or more after the first dose suggested a long detection window of cerumen. Conclusions: Cerumen can be successfully used to detect a single drug ingestion even immediately after the ingestion when a sufficient amount of cerumen is used.
Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.
Designed multitarget ligands are a popular approach to generating efficient and safe drugs, and fragment-based strategies have been postulated as a versatile avenue to discover multitarget ligand leads. To systematically probe the potential of fragment-based multiple ligand discovery, we have employed a large fragment library for comprehensive screening on five targets chosen from proteins for which multitarget ligands have been successfully developed previously (soluble epoxide hydrolase, leukotriene A4 hydrolase, 5-lipoxygenase, retinoid X receptor, farnesoid X receptor). Differential scanning fluorimetry served as primary screening method before fragments hitting at least two targets were validated in orthogonal assays. Thereby, we obtained valuable fragment leads with dual-target engagement for six out of ten target combinations. Our results demonstrate the applicability of fragment-based approaches to identify starting points for polypharmacological compound development with certain limitations.
Lack of efficacy of a partial adenosine A1 receptor agonist in neuropathic pain models in mice
(2021)
Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain.
The bacteriophage ΦX174 causes large pore formation in Escherichia coli and related bacteria. Lysis is mediated by the small membrane-bound toxin ΦX174-E, which is composed of a transmembrane domain and a soluble domain. The toxin requires activation by the bacterial chaperone SlyD and inhibits the cell wall precursor forming enzyme MraY. Bacterial cell wall biosynthesis is an important target for antibiotics; therefore, knowledge of molecular details in the ΦX174-E lysis pathway could help to identify new mechanisms and sites of action. In this study, cell-free expression and nanoparticle technology were combined to avoid toxic effects upon ΦX174-E synthesis, resulting in the efficient production of a functional full-length toxin and engineered derivatives. Pre-assembled nanodiscs were used to study ΦX174-E function in defined lipid environments and to analyze its membrane insertion mechanisms. The conformation of the soluble domain of ΦX174-E was identified as a central trigger for membrane insertion, as well as for the oligomeric assembly of the toxin. Stable complex formation of the soluble domain with SlyD is essential to keep nascent ΦX174-E in a conformation competent for membrane insertion. Once inserted into the membrane, ΦX174-E assembles into high-order complexes via its transmembrane domain and oligomerization depends on the presence of an essential proline residue at position 21. The data presented here support a model where an initial contact of the nascent ΦX174-E transmembrane domain with the peptidyl-prolyl isomerase domain of SlyD is essential to allow a subsequent stable interaction of SlyD with the ΦX174-E soluble domain for the generation of a membrane insertion competent toxin.
Certain electron-rich 1,4-diborabenzene derivatives efficiently activate single, double, and triple bonds and thereby increasingly compete with transition metals in homogeneous catalysis. This review compares the activation of three model substrates (H2, H2C=CH2, CO2) by (i) 9,10-dihydro-9,10-diboraanthracene dianions, (ii) their neutral carbene-stabilized congeners, (iii) 1,3,2,5-diazadiborinines, and (iv) 1,4,2,5-diazadiborinines. Distinct structure-properties relationships become apparent, the most influential factors being (i) the steric demands of the B-bonded substituents, (ii) the charges on the B-doped (hetero)arenes, (iii) charge polarization as a result of additional N-doping, and (iv) the energies and nodal structures of the frontier orbitals. The observed reactions are explained by a transition metal-like activation mechanism. If the two boron atoms are chemically inequivalent, contributions of a B(+I)/B(+III) mixed-valence state determine the observed regioselectivities when polar substrates are added. The lessons learned from the conversions of the model substrates are subsequently used to rationalize the behavior of the B2 heterocycles also toward more sophisticated substrate molecules. Finally, catalytic cycles based on H2- and H−-transfers, hydroboration reactions, and CO2 reductions will be covered.
We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2′-deoxyguanosine to the aptamer domain of the bacterial 2′-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.
We developed three bathochromic, green-light activatable, photolabile protecting groups based on a nitrodibenzofuran (NDBF) core with D-π-A push–pull structures. Variation of donor substituents (D) at the favored ring position enabled us to observe their impact on the photolysis quantum yields. Comparing our new azetidinyl-NDBF (Az-NDBF) photolabile protecting group with our earlier published DMA-NDBF, we obtained insight into its excitation-specific photochemistry. While the “two-photon-only” cage DMA-NDBF was inert against one-photon excitation (1PE) in the visible spectral range, we were able to efficiently release glutamic acid from azetidinyl-NDBF with irradiation at 420 and 530 nm. Thus, a minimal change (a cyclization adding only one carbon atom) resulted in a drastically changed photochemical behavior, which enables photolysis in the green part of the spectrum.
The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.
The genome of the halophilic archaeon Haloferax volcanii encodes more than 40 one-domain zinc finger µ-proteins. Only one of these, HVO_2753, contains four C(P)XCG motifs, suggesting the presence of two zinc binding pockets (ZBPs). Homologs of HVO_2753 are widespread in many euryarchaeota. An in frame deletion mutant of HVO_2753 grew indistinguishably from the wild-type in several media, but had a severe defect in swarming and in biofilm formation. For further analyses, the protein was produced homologously as well as heterologously in Escherichia coli. HVO_2753 was stable and folded in low salt, in contrast to many other haloarchaeal proteins. Only haloarchaeal HVO_2753 homologs carry a very hydrophilic N terminus, and NMR analysis showed that this region is very flexible and not part of the core structure. Surprisingly, both NMR analysis and a fluorimetric assay revealed that HVO_2753 binds only one zinc ion, despite the presence of two ZBPs. Notably, the analysis of cysteine to alanine mutant proteins by NMR as well by in vivo complementation revealed that all four C(P)XCG motifs are essential for folding and function. The NMR solution structure of the major conformation of HVO_2753 was solved. Unexpectedly, it was revealed that ZBP1 was comprised of C(P)XCG motifs 1 and 3, and ZBP2 was comprised of C(P)XCG motifs 2 and 4. There are several indications that ZBP2 is occupied by zinc, in contrast to ZBP1. To our knowledge, this study represents the first in-depth analysis of a zinc finger µ-protein in all three domains of life.
We report here the nuclear magnetic resonance 19F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.
Phytochrome photoreceptors operate via photoisomerization of a bound bilin chromophore. Their typical architecture consists of GAF, PAS and PHY domains. Knotless phytochromes lack the PAS domain, while retaining photoconversion abilities, with some being able to photoconvert with just the GAF domain. Therefore, we investigated the ultrafast photoisomerization of the Pr state of a knotless phytochrome to reveal the effect of the PHY domain and its “tongue” region on the transduction of the light signal. We show that the PHY domain does not affect the initial conformational dynamics of the chromophore. However, it significantly accelerates the consecutively induced reorganizational dynamics of the protein, necessary for the progression of the photoisomerization. Consequently, the PHY domain keeps the bilin and its binding pocket in a more reactive conformation, which decreases the extent of protein reorganization required for the chromophore isomerization. Thereby, less energy is lost along nonproductive reaction pathways, resulting in increased efficiency.
The structure and flexibility of RNA depends sensitively on the microenvironment. Using pulsed electron-electron double-resonance (PELDOR)/double electron-electron resonance (DEER) spectroscopy combined with advanced labeling techniques, we show that the structure of double-stranded RNA (dsRNA) changes upon internalization into Xenopus lævis oocytes. Compared to dilute solution, the dsRNA A-helix is more compact in cells. We recapitulate this compaction in a densely crowded protein solution. Atomic-resolution molecular dynamics simulations of dsRNA semi-quantitatively capture the compaction, and identify non-specific electrostatic interactions between proteins and dsRNA as a possible driver of this effect.