Kaiserslautern - Fachbereich Chemie
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Synergism of Lipoates and Established Anticancer Drugs in Cell and Mouse Models of Colorectal Cancer
(2021)
Das kolorektale Karzinom (KRK) ist eine der am häufigsten auftretenden Krebsentitäten und zeigt aktuell eine erhöhte Inzidenz und Mortalität bei Erwachsenen unter 50 Jahren in Europa und den USA auf. Zumeist im fortgeschrittenen Stadium diagnostiziert, ist die 5-Jahres-Überlebensrate des KRKs immer noch gering. Daher besteht eine Notwendigkeit neuer Therapieansätze und Angriffspunkte für Wirkstoffkandidaten, wenngleich Standardtherapien mit den Zytostatika 5-Fluorouracil (5 FU) oder Irinotecan (IT) und Biologika existieren. Einen solchen Angriffspunkt könnte der Energiemetabolismus darstellen, der für Krebszellen charakteristische Veränderungen aufweist. Das Lipoat CPI 613 ist ein Derivat der natürlich vorkommenden α Liponsäure (LA) und gehört aufgrund seiner einzigartigen Hemmung des veränderten Energiemetabolismus in Krebszellen als Vorreiter zu einer neuen Klasse von Wirkstoffsubstanzen. CPI-613 erwies sich bereits als Inhibitor mitochondrieller Multienzymkomplexe wie der Pyruvatdehydrogenase und der α Ketoglutaratdehydrogenase. Diese Wirkung wurde vorrangig in Krebszellen beschrieben.
Der Fokus dieser Arbeit lag zunächst auf der Untersuchung zellulärer Antworten auf die Behandlung von KRK-Zellen mit dem Lipoat CPI 613 und schlossen neben Effekten auf die mitochondrielle Integrität und Funktion der oxidativen Phosphorylierung auch die Endpunkte Zelltod, DNA-Schädigung und Autophagie ein. Hierfür wurde ein Panel an KRK-Zellen und auch nicht maligne transformierte humane Kolonepithelzellen (HCEC) untersucht. Darüber hinaus wurde in isolierten murinen Mitochondrien die Wirkungsweise von CPI-613 geprüft. Weiterhin sollte ein möglicher Synergismus durch eine Kombinationsbehandlung von Lipoaten wie CPI-613 oder dessen Muttersubstanz LA und Standardchemotherapeutika in der KRK-Behandlung wie 5-FU und IT charakterisiert werden. Nach Identifizierung der aussichtsreichsten Kombination in vitro folgten Studien zur Wirksamkeitssteigerung der Kombinationsbehandlung im Vergleich zur Einzelbehandlung in vivo sowie eine Beurteilung zu möglichen hämatotoxischen Nebeneffekten. Zu diesem Zwecke wurden sowohl das Xenograft-Modell in immundefizienten Mäusen (BALB/c nu/nu) als auch das Azoxymethan (AOM)/Dextran Natriumsulfat (DSS)-Model zur chemischen Induktion von KRK-Tumoren in C57BL/6-Mäusen genutzt.
Es konnte gezeigt werden, dass CPI-613 sowohl in isolierten Mitochondrien als auch in KRK-Zelllinien zu einer Reduktion des mitochondriellen Membranpotentials neben einer vermehrten Bildung von reaktiven Sauerstoffspezies führte. Dies ging mit einer deutlichen Verminderung der zellulären Atmung einher und äußerte sich in KRK-Zelllinien zudem in einer Reduktion der Mitochondrien-Anzahl. Während kein Zellzyklusarrest durch die Behandlung mit CPI 613 ausgelöst wurde, konnte in einem Panel von diversen KRK-Zelllinien Zelltod nachgewiesen werden. Dies war mit gleicher Potenz in den verschiedenen Zelllinien unabhängig des p53- und MSS/MSS-Status zu beobachten. Dabei wurden verschiedene und teils redundante Zelltodmechanismen wie Apoptose, Nekroptose und Caspase-unabhängigem Zelltod nach CPI-613 Behandlung nachgewiesen. Als Folge einer Behandlung mit CPI-613 kam es des Weiteren zu einer gesteigerten Autophagie-Rate in KRK-Zellen. Analysen zum genotoxischen Potential von CPI-613 ergaben keine Hinweise auf DNA-Schädigungen. Verschiedene Kombinationen von Lipoaten und Standardchemotherapeutika wurden in vitro auf deren Synergismus charakterisiert. Neben einer synergistischen Wirkung von CPI-613 in Kombination mit IT in KRK-Zellkulturmodellen konnte ebenfalls ein positiver Effekt in Mausmodellen des KRKs verzeichnet werden. Während CPI-613 bereits alleine in Xenograft-Modellen zu einer Reduktion des Tumorwachstums und somit zu einer verlängerten Überlebenszeit und damit einem Therapieerfolg führte, verstärkten sich diese Effekte deutlich in der Kombinationsbehandlung mit IT. In chemisch-induzierten Tumoren hingegen zeigte vor allem IT einen therapeutischen Effekt, welcher ebenfalls in der Kombination mit CPI-613 zu verzeichnen war. Eine Monotherapie mit CPI 613 führte in diesem Modell zu keinem signifikanten Therapieerfolg. Der Synergismus in vitro und in vivo gründet vornehmlich auf einer gesteigerten Zelltodrate, der Depletion von p53 sowie einer Reduktion der Autophagierate und nicht auf einer erhöhten DNA-Schädigung. Ein hämatotoxisches Nebenwirkungsprofil von CPI-613 wurde hier im Allgemeinen nicht beobachtet.
Zusammenfassend wurde in dieser Arbeit demonstriert, dass CPI-613 im Kontext von KRK den veränderten Energiemetabolismus angreift und zum Zelltod führt. Darüber hinaus konnte eine Genotoxizität von CPI-613 ausgeschlossen werden. Eine Kombination aus CPI-613 und IT führte in Xenograft- und chemisch-induzierten KRK-Tumoren zu einer gesteigerten Wirksamkeit in Bezug auf die Hemmung des Tumorwachstums und der Überlebenszeit. Die Befunde in Zellkultur- und Maus-Modellen des KRKs identifizierten CPI-613 als vielversprechenden Therapiebaustein für die Behandlung des KRKs.
Compared to canonical model organisms, the genetic toolbox of Kinetoplastid parasites have a considerable gap in the transgenic techniques available. The implementation of the CRISPR/Cas9 technology is poised to transform the way we perform genetic manipulations and offers a new and exciting horizon for molecular parasitology. In this study, we use the Kinetoplastid parasite Leishmania tarentolae as a model organism. This unicellular eukaryote is an attractive model for both basic and applied research. Understanding Leishmania’s basic biology is valuable to underpin differences to the host that might help to treat infectious diseases. Furthermore, it also provides new examples of non-conserved mechanisms that will help to understand the fundamental principles of the biology of eukaryotes and their evolution. In this work, the CRISPR/Cas9 system was used to study mitochondrial protein import.
Here I show the efficacy of CRISPR/Cas9 to generate knockout and knockin mutants. Proof- of- concept gene PF16 was used to generate knockout immotile parasites and knockin fluorescent mutants fused with mCherry. The APRT gene was also knocked out showing resistance to APP.
In addition, I generated endogenous mutants of a constituent of the mitochondrial import machineries, the sulfhydryl oxidoreductase Erv. I showed that the KISS domain and cysteine 17 are dispensable for survival dismissing that their functions correlate with the essential operation/s of Erv. I report that the ERV gene and the intervening sequences of its shuttle pair cysteines are refractory to ablation and modification, respectively, indicating that they are essential for survival. I also generated Erv interactomes using full-length and mutant (ErvΔKISS) baits showing candidates with hitherto unknown functions that might be related to Erv function.
I also tested the glmS riboswitch and generate endogenous mutants with CRISPR/Cas9. We asked if it was possible in Leishmania to obtain knockdown mutants with this technique. The evidence of this study indicates that the system is inefficient in provoking a knockdown phenotype for the genes characterized.
An alternative negative marker was also developed in this work. I propose the APRT gene as a novel and efficient counter-selectable marker as compared to the current yFCU and TK genes. The implementation of this system could lead to first shuffling experiments that are not feasible in Leishmania further highlighting the value of this model organism.
Despite their “weak nature” London dispersion interactions are omnipresent and of fundamental importance for many aspects of chemistry and biology and have often been underestimated in the description of intra- and intermolecular interactions. In this thesis, London dispersion is investigated in the gas phase with molecular beam experiments and quantum chemistry. The focus of this work lies in the investigation of London dispersion in the electronic ground state and the electronically excited state. For the electronic ground state, dispersion-bound dimers of triphenylmethane derivatives were analyzed. Depending on the dispersion energy donor, a tail-to-tail (TPM), head-to-tail (iPrTPM) or head-to-head (tBuTPM) arrangement can be assumed for the minimum structure. The tBuTPM dimer exhibits an exceptionally small C-H·· H-C contact which is stabilized by strong London dispersion interactions which was quantified by energy composition analysis. For the characterization of the dimer, the calculation of anharmonic frequencies was of high importance and was also validated with literature data. The second system, the chromone-MeOH balance represents an ideal molecular balance with two competing docking sites at the carbonyl oxygen. The experimental results are compared to theoretical predictions obtained from (TD)DFT-, DLPNO-CCSD(T) and SAPT-calculations to study the balance between electrostatics, induction and dispersion interaction in the S0 and T1 state. The chromone-solvent system was identified as an ideal system for studying London dispersion in multiple electonic states. Furthermore, candidates for derivivatives of chromone were analyzed with quantum chemical methods in the electronic ground and electronically excited state in an attempt to identify suitable candidates for further experiments. The 6-methylchromone shows promising behavior in stabilizing the inside pocket regardless of the electronic state and was analyzed in more detail with a variety of methods. Similar analysis of 2-CF3chromone and the 2-CF3, 6-methylchromone showed no special effect of a substitution in 2-position or possible cooperative effects.
Pyrrolizidine alkaloids (PA) are secondary plant metabolites occurring in a great many plant species worldwide, known to exhibit hepatotoxic, genotoxic and carcinogenic properties after metabolic activation. In recent years, contamination of food, feed and herbal medicines with PA has become an increasing problem. The concept of interim relative potency factors (iREP) proposed by Merz and Schrenk in 2016 was a new approach for risk assessment of PA. While existing approaches of risk assessment assumed equivalent toxic potency for all PA congeners, the approach of Merz and Schrenk considered the structural features of individual PA congeners based on existing data from the literature. In order to generate further data on the structure-specific toxicity of PA, congeners of different structural classes were investigated in different in vitro test systems.
In vitro cytotoxicity was investigated in primary rat hepatocytes, HepG2 C9 cells (overespressing human CYP3A4) and naïve HepG2 cells. Overall, it could be observed that lasiocarpine and the cyclic di-esters (except monocrotaline) showed much stronger cytotoxic effects in comparison to the tested mono-esters in both, primary rat hepatocytes and in HepG2 C9 cells. Primary hepatocytes were the most sensitive cells investigating cytotoxicity of different PA congeners, followed by the HepG2 C9 cells. This is confirmed by markedly higher metabolism rates for all investigated PA in primary rat hepatocytes determined in the metabolism experiments. In naïve HepG2 cells no cytotoxic effects could be observed. The influence of cytochrome P450 (CYP) on the formation of toxic metabolites seem to play a crucial role. This assumption could be beared using ketoconazole as CYP inhibitor and testing various pre-incubation times in primary rat hepatocytes. CYP activity was measured using 7-Benzoxyresorufin-O-Dealkylase (BROD) assay in primary rat hepatocytes and in HepG2 C9 cells. Glutahione (GSH) depletion using buthionine sulfoximine (BSO) showed slight stronger cytotoxic effects for several PA, but not for all tested.
In contrast to the negative results of mutagenicity in ames fluctuation assay using Salmonella strains TA98 and TA100 with and without metabolic activation by S9 mix, all tested PA congeners showed micronuclei induction in the HepG2 C9 cell line. Again, laisocarpine and the cyclic di-esters (except monocrotaline) were the most potent ones. In conclusion, the data from cytotoxicity and genototoxicity experiments from the tested PA congeners confirm published iREP factors with a few exceptions, in particular for monocrotaline or echimidine.
Additionally, metabolism of six selected PA was studied in primary rat hepatocytes and HepG2 C9 cells. Genrally, it was found that almost all tested cyclic and open-chained di-esters (except retrorsine) showed much higher metabolism rates in both cell types, in comparison to the mono-esters, for which only low metabolism rates could be measured. The same was observed for the quantified amounts of reactive metabolites in the supernatants of both cell types. In general, also these data bear the results from cytotoxicity and genotoxicity experiments and help to better understand the complex metabolism and the structure-specific toxicity of different PA congeners.
This thesis is separated into seven distinct research projects on mono and multinuclear transition metal complexes as trapped ions in gas phase, as well as one chapter on focusing on the development of a new ion source to enable access to catalytic processes via coadsorption.
ElectroSpray Ionization (ESI) transfers ions from solution to gas phase for mass spectrometric investigations, allowing a broad variety of experimental methods to obtain fundamental insights into the molecular properties of isolated complexes devoid of solvent, crystal lattice, bulk, or supporting surface effects.
Collision Induced Dissociation (CID) researches molecular fragmentation mechanisms and their relative gas phase stabilities at room temperature. Laser experiments such as InfraRed (Multiple) Photon dissociation and Ultraviolet Photon dissociation offer information on the bonding motifs, resulting in molecular structures and their electronic ground states. When quantum chemical calculations utilizing Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TD-DFT) are combined with monitored spectra, a better and deeper understanding of the structural properties and electronics of transition metal complexes is possible.
X-ray magnetic circular dichroism (XMCD) is a technique that analyzes the magnetic properties of isolated and trapped ions at cryogenic temperatures inside an externally applied magnetic field using high brilliant polarized X-ray photons in conjunction with a mass spectrometer. The element selective technique, combined with sum analysis, allows for the decomposition of the total magnetic moments in their spin and orbital magnetic moments in various metal centers. A determination of the magnetic couplings between distinct metal centers in multinuclear complexes is possible via Broken symmetry approach in combination with X-ray Magnetic Circular Dichroism (XMCD).
Qualitative NMR spectroscopic and quantitative calorimetric binding studies were performed to characterize the interaction of nontoxic mimics of the V-type nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) and the Novichok nerve agent A-234 (ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate) with a series of receptors in 100 mM aqueous phosphate buffer at pH 7.4 and 37°C. These investigations provided information about the preferred geometry with which the nerve agent mimics are included into the receptor cavities and about the stability of the complexes formed. According to the results, the positively charged VX mimic prefers to bind to cation receptors such as sulfonated calixarenes and an acyclic cucurbituril but does not noticeably interact with cyclodextrins. While binding to the acyclic cucurbituril is stronger than that to calixarenes, the mode of inclusion into the sulfonatocalix[4]arene cavity is better suited for the development of scavengers that bind and detoxify V-type nerve agents. The neutral Novichok mimic, on the other hand, only interacts with the acyclic cucurbituril with a strength required for scavenger development. These binding studies thus provided guidelines for the further development of nerve agent scavengers.
Within toxicology, reproductive toxicology is a highly relevant and socially particularly sensitive field.
It encompasses all toxicological processes within the reproductive cycle and therefore includes many effects and modes of action. This makes the assessment of reproductive toxicity very challenging despite the established in vivo studies. In addition, the in vivo studies are very demanding both in terms of their conduct and interpretation, and there is scope for decision-making on both aspects. As a result, the interpretation of study results may vary from laboratory to laboratory. For the final classification, the assessment of relevance for men is decisive. The problem here is that relatively little is known about the species differences between men and the
usual test animals (rat and rabbit). The rabbit in particular has hardly been researched in molecular biology. The aim of the dissertation was to develop approaches for a better assessment of
reproductive toxicity, with two different foci: The first aim was to investigate species differences, focusing on the expression of xenobiotic transporters during ontogeny. Xenobiotic transporters, of the superfamily of ATP-binding cassette transporters (ABC) or solute carriers (SLC), are known to transport exogenous substances in
addition to their endogenous substrates and therefore play an important role in the absorption, distribution and excretion of xenobiotics. Species differences in kinetics can in turn have a major
impact on toxic effects. In the study, the expression of 20 xenobiotic transporters during ontogeny was investigated at the mRNA level in the liver, kidney and placenta of rats and rabbits and compared with that of men. This revealed major differences in the expression of the transporters between the species. However, further studies on the functionality and activity of the xenobiotic transporters are needed to fully assess the kinetic impact of the observed species differences. Overall, the study provides a valid starting point for further systematic investigations of species differences at the protein level. Furthermore, it provides previously unavailable data on the expression of xenobiotic transporters during ontogeny in rabbits, which is an important step in the molecular biological study of this species.
The second part focused on investigating the predictive power of in silico models for reproductive
toxicology in relation to pesticides. Both the commercial and the freely available models did not
perform adequately in the evaluation. Three reasons could be identified for this: 1. many pesticides
are outside the chemical space of the models, 2. different definition/assessment of reproductive
toxicity and 3. problems in detecting similarity between molecules. To solve these problems, an
extension of the databases on reproductive toxicity in relation to pesticides, respecting a uniform
nomenclature, is needed. Furthermore, endpoint-specific models should be developed which, in
addition to the usual structure-based fingerprints, use descriptors for, for example, biological
activity.
Overall, the dissertation shows how essential it is to further research the modes of action of
reproductive toxicity. This knowledge is necessary to correctly assess in vivo studies and their
relevance to men, as well as to improve the predictive power of in silico models by incorporating
this information.
Wine is a complex chemical mixture that is bound to change over time. Most wines are
produced for consumption within months. Some premium wines are meant to be maturated for
several years or even decades after bottling. The post-bottling evolution and the longevity of a
wine depends on its initial chemical composition and the storage conditions. Temperature,
exposure to light and the closure type are often mentioned as the most important storage
influences. Especially elevated temperature is known to cause accelerated aging reactions in
wine. Refrigerated wine storage cabinets promise to be the best storage option without the
need of a wine cellar. They are available in different sizes and fit in every household. However,
the influence of vibrations and low-interval temperature fluctuations caused by compressors
are parameters that have been neglected in literature. The aim of this thesis was to investigate
if vibrations and low-interval temperature fluctuations, which occur in refrigerated wine storage
cabinets, have an influence on the post-bottling evolution of a wine. The influence of both
parameters was studied separately from each other.
The impact of vibration on oxidation and gas uptake from the headspace of a wine bottle into
the wine was investigated using a model wine with saturated O2 and different headspace
volumes. The study revealed that vibration promotes the dissolution of O2 from the headspace
of bottle into the wine resulting in a faster SO2 consumption. Furthermore, it was shown that
horizontal bottle position accelerated the O2 uptake significantly. It was concluded that the
increased surface size between headspace and wine accelerates the O2 dissolution in wine.
Also, bigger headspace volumes caused an accelerated O2 uptake into the wine. An
experiment without any headspace volume revealed that the factors vibration and bottle
position did not accelerate the O2 consumption in wine. This proves that vibration and bottle
position accelerate only the dissolution of O2 in wine, but not the chemical reaction of O2 with
wine constituents.
The influence of vibration on the volatile profile of wine was investigated using Riesling
sparkling and still wines sealed with different closures that were subjected to vibration for six
months. Vibration caused no CO2 losses, SO2 and color changes in all wines indicating that
vibration caused by compressors has no impact on the gas permeability of the used closures.
However, vibration affected the volatile profile of sparkling wine and Riesling still wine sealed
with a screw cap. Similar to the model wine study described earlier, it was shown that the
equilibrium of volatile substances between the wine and the headspace in a bottle was
influenced by vibration. The gas-liquid-equilibrium of some volatile compounds was shifted
towards wine, while others were shifted towards headspace. As a result of this, the
concentration of volatile compounds in wine is changed. Besides this indirect influence of
vibration, the results of this study also suggested that specific degradation and formation
reactions of volatile compounds are directly influenced by vibration. These multiple effects of
2
vibration most likely explain why increasing vibration intensities could not be proportionally
related to the observed volatile changes. The investigation of different wine styles revealed
that the impact of vibration depends strongly on the initial composition of wine, age, and
packaging conditions. Especially, headspace volume, closure type and CO2 pressure are likely
to influence the equilibrium of volatile substances between the wine and the headspace in a
bottle.
Another study investigated the impact of low-interval temperature fluctuations on the volatile
profile of wine. For this purpose, a Riesling wine was stored for two years under different
temperature fluctuation patterns caused by compressors. Additionally, a model wine with nine
volatile substances with known concentrations was stored for eight months under the same
fluctuation patterns. The low-interval temperature fluctuations were compared to the mean
value of the temperature fluctuations. Chemical and sensory analysis revealed that that lowinterval temperature fluctuations accelerate wine aging reactions like ester hydrolysis and
monoterpene degradation. Even small temperature amplitudes showed a significant impact on
wine aging. The observed effect was explained by the Arrhenius equation which states that
reaction rates exponentially increase with rising temperatures. A pump effect of air through the
closure was initially assumed but not observed in this study. Small deviations in wine
temperature, such as those caused by door openings of a refrigerator were found to be
negligible. It was concluded that low-interval temperature fluctuations can accelerate wine
aging reactions. The amplitude of the temperature fluctuations should be as small as possible
during bottle storage of wine.
This thesis showed that both parameters, vibration, and low-interval temperature fluctuations,
have been proven to influence the evolution of wine during bottle storage. Regarding storage
conditions in a refrigerated wine storage cabinet, those parameters should be monitored. Wine
connoisseurs should therefore consider good wine cabinets, since some manufacturers
emphasize on the importance to minimize vibrations and temperature fluctuations in their
devices. The development of technology should be advanced to reduce both vibration and
temperature fluctuations in refrigerated wine storage cabinets. Future research should focus
on specific wine compounds in model systems and realistic vibration conditions to reveal the
relationship between vibration intensities and reaction rates. The impact of low-interval
temperature fluctuations on wine compositional changes should be investigated considering
horizontal and vertical bottle positions. The calculated acceleration factors due to temperature
fluctuations have to be verified by isotherm storage conditions at higher temperatures.
Hordatines are a characteristic class of secondary metabolites found in barley which have
been reported to be present in barley malt, beer and, recently, brewer ́s spent grain (BSG). However,
little is known about their biological activities such as antioxidative effects in beer or antifungal
activity as their main task within the plants. We conducted an in vitro investigation of the activity
of hordatines isolated from BSG towards enzymes of glucose metabolism. Hordatine-rich fractions
from BSG were prepared by solid-liquid extraction (SLE) with 60% acetone followed by purification
and fractionation. The fractions were characterised and investigated for their in vitro inhibitory
potential on α-glucosidase and glycogen phosphorylase α (GPα). Both enzymes are relevant within
the human glucose metabolism regarding the digestion of carbohydrates as well as the liberation of
glucose from the liver. In total, 10 hordatine-rich fractions varying in the composition of different
hordatines were separated and analysed by mass spectrometry. Hordatine A, B and C, as well as
hydroxylated aglycons and many glycosides, were detected in the fractions. The total hordatine
content was analysed by HPLC-DAD using a semi-quantitative approach and ranged from 60.7 ± 3.1
to 259.6 ± 6.1 μg p-coumaric acid equivalents/mg fraction. Regarding the biological activity of
fractions, no inhibitory effect on GPα was observed, whereas an inhibitory effect on α-glucosidase
was detected (IC50 values: 77.5 ± 6.5–194.1 ± 2.6 μg/mL). Overall, the results confirmed that
hordatines are present in BSG in relatively high amounts and provided evidence that they are potent
inhibitors of α-glucosidase. Further research is needed to confirm these results and identify the active
hordatine structure.
Work on the use of cyclic peptides or pseudopeptides as synthetic receptors started even before the field of supramolecular chemistry was firmly established. Research initially focused on the development of synthetic ionophores and involved the use of macrocycles with a repeating sequence of subunits along the ring to facilitate the correlation between structure, conformation, and binding properties. Later, nonnatural amino acids as building blocks were also considered. With growing research in this area, cyclopeptides and related macrocycles developed into an important and structurally diverse receptor family. This review provides an overview of these developments, starting from the early years. The presented systems are classified according to characteristic structural elements present along the ring. Wherever possible, structural aspects are correlated with binding properties to illustrate how natural or nonnatural amino acids affect binding properties.