Lehrstuhl für Umweltchemie und Analytische Chemie

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Transcriptomics-based developmental toxicity and cardiotoxicity assessment using induced pluripotent stem cells
(2023) Cherianidou, Anna; Hengstler, Jan G.; Sachinidis, Agapios
This PhD study focuses on developing an innovative in vitro test system using human-induced pluripotent stem cells (hiPSCs) to assess developmental toxicity. Traditional approaches rely on animal studies, which can be complex, costly, and less applicable to humans. The study introduces the UKK2 protocol combined with hiPSCs and transcriptomics to evaluate both developmental teratogenicity and cardiotoxicity. The research involves differentiating hiPSCs into various germ layers and cardiomyocytes in the presence of substances known to have embryotoxic effects. Gene expression analyses identify gene signatures associated with early embryotoxicity and cardiotoxicity. The study's first phase assesses the effectiveness of the UKK2 test system with 23 teratogens and 16 non-teratogens at varying concentrations. A classifier with high accuracy (90% to 92%) is established for predicting teratogens using the UKK2 test system. The research extends the UKK2 protocol to evaluate developmental cardiotoxicity (UKK-CTT). A gene signature predicts the inhibitory potential of teratogens and non-teratogens in cardiomyogenesis. The "Developmental Cardiotoxicity Index" (CDI31g) effectively distinguishes compounds affecting hiPSC differentiation into functional cardiomyocytes. This study anticipates a promising future for human-based in vitro models in developmental toxicity screening, improving drug testing and teratogenicity prediction. These advancements provide valuable insights into drug-induced developmental toxicity during cardiac organogenesis, enhancing our understanding of critical embryonic development phases and drug safety during vulnerable periods.
Transcriptomics-based identification of teratogens in differentiating hiPSCs: A novel in vitro test system
(2021) Seidel, Florian; Hengstler, Jan; Watzl, Carsten
The knowledge about the developmental toxicity potential of substances is a matter of high concern in toxicology and medicine, especially if pregnant women need to take potentially teratogenic drugs to treat severe infections or diseases. The regulatory risk assessment of such substances is, however, very difficult, costly and time-consuming and relies currently, due to a lack of appropriate alternatives, solely on animal studies. During the last decades, much effort was put into the development and establishment of trustworthy in vitro-based test systems for the risk assessment of teratogenic substances, but not even one was approved for this purpose yet. In this PhD project, a new attempt was undertaken to develop such a system. The here presented novel system is based on the UKN1 assay, in which pluripotent human embryonic stem cells were differentiated to neuroepithelial precursor cells, and made considerable innovations to it by utilizing human induced pluripotent stem cells (hiPSCs), whole-transcriptome analyses and a thoroughly chosen set of 39 teratogenic and non-teratogenic substances that each were applied at two on human in vivo data-based concentrations to differentiating hiPSCs in this “UKN1 6-day” assay. In contrast to the non teratogenic compounds, the teratogens induced either significant gene expression alterations or a high toxicity in almost all cases, so that this test system was able to predict substances with a high accuracy of 90 %, a robust sensitivity of 83 % and an outstanding specificity of 100 %, what makes it therefore to a very high performing in vitro test system. Even a variation of the system, the UKN1 one-day assay, which shortened the total time requirements of the assay and replaced the whole-transcriptome analyses by a four biomarker-based RT-qPCR, could predict the compounds with an accuracy, sensitivity and specificity of 77 %, 63 % and 100 %, respectively. In addition to the core set of 39 substances, the UKN1 6-day assay was used to determine the health risks of parabens which are widely used as preservatives in cosmetics. No significant gene alterations were found in differentiated, paraben-exposed hiPSCs, and the obtained negative in vitro results could now support the existing in vivo data of paraben-exposed animals in accordance with a read-across-approach for ethylparaben, so that no further animal experiments would be needed for the regulatory risk assessment of ethylparaben. In the near future, steps for a further optimization of the test system will be investigated like the application of targeted instead of whole-transcriptome analyses and multiple concentrations instead of just two. Furthermore, the number of tested compounds shall be increased. Although there is still a long way to go and many hurdles to overcome until this or another test system will be approved for the regulatory risk assessment of teratogens, the UKN1 6-day test system can already provide a tool for an in vitro teratogenicity screening of compounds and aid authorities and companies to assess the health risks of potentially teratogenic substances.
Development of an in vitro test battery for a test system to predict human drug-induced liver injury
(2021) Brecklinghaus, Tim; Hengstler, Jan G.; Rahnenführer, Jörg
Drug-induced liver injury (DILI) is a major concern due to its poor predictability. Recently, we have developed an in vitro/in silico test system for the prediction of human DILI in relation to oral doses and blood concentrations. Additionally, two indices, the toxicity separation index (TSI) and the toxicity estimation index (TEI) were introduced for the quantitative evaluation of a test system and its input parameters. In this PhD-thesis, I studied whether extending the in vitro test battery of the test system, so far consisting of a cytotoxicity test in primary human hepatocytes, by additional functional readouts would lead to improved performance and thus allow a more accurate prediction. In total, three different approaches that address putative DILI-relevant mechanisms were explored. In the first approach, the influence of a bile acid mix on the cytotoxicity of in total 18 compounds in cultivated primary human hepatocytes was investigated. In summary, increased and decreased susceptibility to both hepatotoxic and non-hepatotoxic substances was observed with the addition of bile acids, which did not improve the TSI (0.79 -> 0.77) nor the TEI (0.73 -> 0.69) compared to cytotoxicity without additional bile acids. In the second approach, an assay was developed and evaluated that measures the inhibition of bile acid export carriers in primary human hepatocytes. In total 36 compounds were tested with the transport inhibtion assay and the cytotoxicity assay. In conclusion, the assay is able to detect bile acid export carrier inhibition and integration into the in vitro test battery improved TSI (0.77 -> 0.89) and TEI (0.69 -> 0.83) compared to cytotoxicity. In a third approach, intracellular lipid accumulation in HepG2 cells was investigated for a total of 60 compounds. Addition of lipid droplet accumulation to cytotoxicity improved TSI (0.74 -> 0.80) and TEI (0.67 -> 0.81). In summary, three assays were developed for the in vitro test battery of a test system to predict drug-induced liver injury. Quantitative analysis revealed that two of the three assays lead to improved separation of hepatotoxic and non-hepatotoxic compounds, as well as improved estimation of in vivo relevant blood concentrations. These improvements allow more accurate prediction of DILI by the test system.
Supplement to: Multi-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
(2021) Nell, Patrick; Hengstler, Jan G.; Rahnenführer, Jörg
Multi-OMICS analysis of stem cell-derived hepatocyte-like cells reveals a liver-intestine hybrid state that can be targeted by bioinformatics-guided interventions
(2021) Nell, Patrick; Hengstler, Jan G.; Rahnenführer, Jörg
Humane, induzierte pluripotente Stammzellen (iPSC) zeigen Potenzial für die therapeutische Applikation in der personalisierten Medizin, sowie die Verwendung zur Entwicklung von Modellsystemen in pharmakologischen und toxikologischen Studien. Die Differenzierung von iPSC zu Hepatozyten-ähnlichen Zellen wurde durch eine Vielzahl an Protokollen realisiert, wobei die resultierenden Zellen ein variierendes Ausmaß phänotypischer Reife aufweisen. Die genomweite Expression zeigt enorme Unterschiede zu primären Hepatozyten (PHH), veranschaulicht an Hand der Expression von Leber- wie auch Darm-assoziierten Genen. In dieser Arbeit wurde die Differenzierung von iPSC über definitive Endoderm (DE) zu HLC mittels Einzelzell- sowie Bulk-RNA-Sequenzierung und epigenetischen Analysen charakterisiert. Für die Auswertung wurde eine überwachte Strategie zur Gruppierung von Genen mit ähnlichen Expressionsverläufen in Differenzierungsmuster-Gruppen (DPG) entwickelt. Diese DPGs wurden mit bioinformatischen Methoden analysiert um Gen-Netzwerke und Regulatoren zu identifizieren, die zu gewünschter oder auch unerwünschter Differenzierung beitragen. Epigenetische Analysen konnten zeigen, dass eine Verbindung zwischen der Zugänglichkeit von Chromatin und der Expression erwünschter und unerwünschter Faktoren besteht und dass HLC im Vergleich zu PHH eine globale hyper-Methylierung ihrer Promoterregionen aufweisen. Dies legt grundsätzlich nahe, dass HLC nicht die für Hepatozyten charakteristische epigenetische Landschaft ausbilden. Außerdem konnte mittels Einzelzell-Sequenzierung gezeigt werden, dass HLC eine einzige Zellpopulation darstellen, welche in einem Hybridzustand verharrt, in dem Hepatozyten-assoziierte Gene zusammen mit unerwünschten – hauptsächlich Darm-assoziierten - Genen, die weder in adulten noch fetalen primären Hepatozyten vorkommen, exprimiert werden. Diese Ergebnisse verdeutlichen, dass in der Differenzierung von HLC in vitro wichtige Signale fehlen, die der Differenzierung von Hepatozyten in vivo zu Grunde liegen. Analysen zur Überrepräsentation von Gen-Regulatoren, die zwischen HLC, fetalen und adulten primären Hepatozyten unterschiedlich exprimiert werden, deuteten auf eine zentrale Rolle für den Kernrezeptor FXR hin. Die Kombination von FXR Expression und Aktivierung des Rezeptors durch Agonisten konnte die Expression von Hepatozyten-assoziierten Genen fördern, während die Expression unerwünschter Darmgene teils unterdrückt wurde. Auf diese Weise konnte gezeigt werden, wie die HLC Differenzierung durch gezielte Manipulation von Gennetzwerken verbessert werden kann.
Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations
(2021) Albrecht, Wiebke; Hengstler, Jan G.; Watzl, Carsten
Drug-induced liver injury (DILI) is the leading cause for acute liver failure in the USA and in Germany and one of the most common reasons for withdrawal of drugs from the market or failure of a drug candidate during development. Since DILI cannot be accurately predicted by animal models, a reliable in vitro test system for the prediction of human hepatotoxicity would be a valuable asset for drug development as well as for regulatory purposes. In this thesis an in vitro/in silico approach for the prediction of human hepatotoxicity in relation to blood concentrations and oral doses was established. This approach combines in vitro effective concentrations derived from a cytotoxicity assay, in vivo concentrations obtained by physiologically based pharmacokinetic (PBPK) modelling and a support vector machine (SVM) classifier based on these concentration pairs to predict the risk for hepatotoxicity for specific exposure scenarios. For systematic test system evaluation and optimization two novel performance metrics, the Toxicity Separation Index (TSI) and Toxicity Estimation Index (TEI), were utilized. These indices eliminate the need for a priori defined in vitro and in vivo concentrations and foster the systematic evaluation of the benefit of additional readouts. As a first step the feasibility of the in vitro/in silico approach was tested for primary human hepatocytes (PHH) and a training set of 28 compounds with in total 30 different in vitro/in vivo concentration vectors, yielding a sensitivity of 100%, a specificity of 88% and an accuracy of 93% in the leave-one-out classification with the SVM based classifier. A SVM based classifier utilizing all vectors was then applied to derive in combination with reverse PBPK modelling an acceptable daily intake (ADI) for the hepatotoxicant pulegone. The derived ADI was comparable to published ADIs based on two rodent studies. Next, the compound set was extended to a total of 80 compounds with 82 distinct in vitro/in vivo concentration pairs. The SVM leave-one-out classification resulted in a sensitivity of 77.8%, a specificity of 59.4% and an accuracy of 70.1%. Furthermore, the feasibility of the approach substituting HepG2 cells for the PHH and a combination of both cell culture systems for the extended compound set was evaluated. The obtained sensitivity was 88.9% and 86.7% and the specificity 62.5% and 65.6%, respectively. The accuracy was in both cases 77.9%.
A role of glycerophosphodiesterase EDI3 in glycogen metabolism
(2020) Leonhardt, Gregor M.; Hengstler, Jan Georg; Watzl, Carsten
The glycerophosphodiesterase EDI3 (endometrial differential 3) is a key enzyme in various metabolic signalling pathways because it cross-links the triglyceride and choline signalling pathway by hydrolysing glycerophosphocholine (GPC) to glycerol-3-phosphate (G3P) and choline. The enzyme has two functional domains - the GDE domain, where the active site of the protein is located, and the carbohydrate binding domain (CBM), whose function is completely unknown. EDI3 is part of the evolutionary conserved GDE protein family, whose members have different functions. In previous work, high EDI3 expression in primary toumors of ovarian and endometrial carcinomas was associated with increased metastasis, increased cell migration and poorer survival. In this work the role of the CBM20 domain was investigated on a large scale and a possible new role for EDI3 was found. The present work confirms previous research on other CBM20 proteins (Laforin and STBD1) that showed that the CBM domain is essential for protein stability. The deletion of the entire CBM20 domain (ΔCBM20) and mutations of a conserved amino acid within the CBM20 (W32A) significantly reduced protein stability. As a consequence of these mutations, the enzymatic activity of the protein was decreased (W32A) or completely eliminated (ΔCBM20) and the phenotypic migration effect was reduced. Furthermore, this work could show that EDI3 forms dimers or oligomers and the CBM20 domain is required for this process. A characteristic of CBM20 is the interaction with plant starch. For laforin and STBD1, an interaction with glycogen via the CBM20 domain was additionally demonstrated. In this work EDI3 could be associated for the first time with glycogen in vitro and with glycogen-associated proteins of the human skeletal and cardiac muscle. While no binding of EDI3 and glycogen could be detected in human liver, glucagon regulation was observed in primary mouse hepatocytes, potentially linking EDI3 to a role during gluconeogenesis. In skeletal muscle, EDI3 was associated with Type II skeletal muscle cells and the T-tubules, where it co-localizes with ryanodine receptor 1, suggesting a role of EDI3 in calcium signalling.
Role of WNT1-inducible signaling pathway protein-1 (WISP1) in liver injury
(2020) González Leiva, Daniela Fernanda; Hengstler, Jan G.; Watzl, Carsten
Liver diseases are a global burden and a better understanding of factors controlling disease progression is required. In the last decade, much progress has been achieved in understanding the relevance of the extracellular matrix. Alterations in this dynamic structure can either facilitate or impair the repair of damaged liver tissue. Therefore, matricellular proteins of the CCN family, have emerged as new targets in liver pathophysiology. These highly conserved secreted proteins specifically interact with and signal through various extracellular partners, like integrins, which enable them to play crucial roles in various processes including development, wound healing and diseases such as cancer and fibrosis. We have discovered that WISP1 (Wnt1-inducible signaling pathway protein-1) also named CCN4, is induced upon CCl4-induced liver damage and may play an important role in the remodeling process of the extracellular matrix. Therefore, the aims were to identify the cell type that produces WISP1, to study its influence in cell migration and to use WISP1 KO mice to understand its role in the pathogenesis of liver fibrosis. Isolation of individual liver cell types and quantification of WISP1 expression and secretion showed a higher mRNA expression and TGF-β-induced secretion of WISP1 in non-parenchymal cells, especially in stellate cells compared to hepatocytes. Furthermore, WISP1 facilitated the migration of isolated mouse hepatic stellate cells through collagen lattices, suggesting the interaction of WISP1 with one of the main components of the extracellular matrix and affecting its architecture. Additionally, gene expression analysis and Sirius Red staining showed differences in the development of CCl4-induced fibrosis between WISP1 wild type and knockout mice. Upregulation of collagen type I and α-SMA is reduced in WISP1 KO mice and less collagen deposition is also observed. In conclusion, WISP1 is mainly expressed and secreted by stellate cells which may influence their migration upon liver injury and consequently, ameliorating the degree of liver fibrosis.
Alterations of the glyoxylate metabolism in hepatic steatosis – a risk factor for hyperoxaluria
(2020) Jashari, Adelina; Hengstler, Jan Georg; Watzl, Carsten
Non-alcoholic fatty liver disease (NAFLD) comprises a wide spectrum of liver diseases and is the leading cause of liver diseases worldwide. Features of the metabolic syndrome and extrahepatic diseases, like cardiovascular diseases and kidney stone disease, strongly associate with NAFLD. A previously identified steatosis-associated downregulation of the alanine-glyoxylate aminotransferase (AGXT) was suggested to be one molecular link explaining the connection of NAFLD with the development of calcium oxalate stones. AGXT is responsible for the hepatic glyoxylate detoxification and its deficiency results in primary hyperoxaluria type 1 (PH1), which is a hereditary disorder leading to the formation of calcium oxalate kidney stones. Furthermore, the AGXT downregulation in steatotic conditions was proposed to be influenced by DNA methylation since the AGXT promoter is hypermethylated in steatosis. In the course of this thesis, further alterations of the glyoxylate metabolism were revealed in the ob/ob and Western diet (WD) mouse models of NAFLD contributing to a better understanding of the steatosis-associated modifications. These alterations indicating an increased hepatic oxalate production in steatotic conditions were confirmed by elevated oxalate excretion of steatotic hepatocytes from WD-fed and ob/ob mice upon hydroxyproline exposure. Furthermore, the increased oxalate production in the fatty liver was validated in vivo by elevated oxalate levels in the plasma from the hepatic vein of WD-fed mice. Additionally, oxalate levels in the plasma and urine of ob/ob mice were increased to a higher extent by dietary hydroxyproline compared to ob/+ mice, showing an enhanced susceptibility towards hydroxyproline also in vivo. The selective sensitivity of steatotic hepatocytes to hydroxyproline contrasted with that of primary hepatocytes from Agxt knock-out (Agxt-/-) mice, which excreted more oxalate when exposed to all oxalate precursors than hepatocytes from wild type control mice. This suggested, that only mitochondrial glyoxylate detoxification is compromised in hepatic steatosis. Rescuing the Agxt expression in the hepatocytes of ob/ob mice by AAV-mediated gene transfer was able to reduce oxalate production from hydroxyproline. Moreover, inhibition of hydroxyproline catabolism normalised the oxalate excretion from ob/ob hepatocytes after consumption of hydroxyproline. These results provided clear evidence that the downregulation of Agxt is, at least partially, responsible for the steatosis-accompanied increased hepatic oxalate production in NAFLD mouse models. This supported the hypothesis of the steatosis-associated downregulation of Agxt being a molecular link explaining the strong association between NALFD and kidney stone disease. The translational relevance of these findings was studied in a cohort of overweight and obese children and adolescents with biopsy proven NAFLD and corresponding 24 h urine samples. The steatosis percentage positively correlated with the amount of urinarily excreted oxalate, showing the relevance of steatosis induced hyperoxaluria in human NAFLD. In order to further understand the deregulation of Agxt expression in steatosis and the dependency to DNA methylation, its transcriptional upregulation in response to glucagon was studied in ob/ob and 6 weeks WD-fed mice. When treated with glucagon, an increase of the Agxt mRNA expression was missing in ob/ob mice but not in 6 weeks WD-fed mice, most probably due to the higher hypermethylation of the Agxt promoter in ob/ob mice. This supported the importance of the Agxt promoter hypermethylation regarding the regulation of the Agxt gene expression. These results were confirmed in vitro in glucagon stimulated primary hepatocytes from the two different NAFLD mouse models and in an in vitro steatosis model. Furthermore, the phosphorylation of Creb was lost earlier in hepatocytes from ob/ob, 6 weeks WD-fed mice and in an in vitro steatosis model. All in all, these findings support the thesis that steatosis associated AGXT promoter hypermethylation might repress the AGXT gene expression leading to an impaired response towards glucagon.
Anwendung der hochaufgelösten bildgebenden MALDI-MS zur Aufklärung der räumlichen Verteilung des Acetaminophens und seiner Biotransformationsprodukte sowie von Taurocholaten in der Leber von Mäusen in Folge einer Acetaminophen Intoxikation
(2019) Sezgin, Selahaddin; Spiteller, Michael; Hengstler, Jan Georg
Überdosierungen an APAP führen zu hepatotoxischen Auswirkungen in der Leber von Säugetieren. Die Hepatotoxizität ist u.a. eine Folge der metabolischen Aktivierung des Acetaminophens durch CYP. Dabei treten pericentrale Schadensmuster in den Leberläppchen auf. Es fehlten bisher Informationen über die räumlich-zeitliche Verteilung vom APAP und seinen Biotransformationsprodukten in der Leber bis zur Veröffentlichung von Teilen dieser vorliegenden Arbeit. Um die Fragestellung zu beantworten, wurde eine Methode basierend auf der leistungsstarken HR-MALDI-MSI Technik entwickelt. Dadurch konnte die Verteilung vom APAP, seinen Hauptmetaboliten APAP-GLC und APAP-SUL und seinem Addukt APAP-GSH in der Leber von C57BL/6 Mäusen, denen APAP intraperitoneal verabreicht wurde und deren Leber sowohl zeit- als auch dosisabhängig beprobt wurden, detektiert werden. Zuvor wurden intensive Anstrengungen unternommen, um das toxische Intermediat NAPQI im Lebergewebe durch MALDI-MSI zu erfassen. Ziel war es, Informationen über seinen Bildungs- und Wirkungsort im Lebergewebe zu gewinnen. Hervorzuheben ist dabei ein Versuchsansatz bei dem H2S zur Derivatisierung von NAPQI eingesetzt wurde. Diese innovative Methode könnte zudem das Portfolio an Derivatisierungsmöglichkeiten für MALDI-MSI Experimente erweitern. Allerdings konnte das NAPQI weder direkt noch über seine artifiziellen Derivate im Lebergewebe detektiert werden, was vermutlich auf einen Mangel an nicht-abreagiertem NAPQI im Lebergewebe zurückzuführen ist. Die durch Gewebehistologie unterstützte Superpositionierung von Ionendichtebildern unter Einbeziehung der Eigenschaften verschiedener Imaging Software ermöglichte es, präferentielle Verteilungen der Analyten bezüglich der metabolischen Zonen zu erfassen. Während für APAP, APAP-SUL und APAP-GLC keine zonale Verteilung festgestellt werden konnte, konnte eine präferentielle Bildung des APAP-GSHs sowie ein schwerpunktmäßiger Abbau des GSHs in der pericentralen Zone verortet werden. Der Abbau an GSH war aber nicht ausschließlich auf die pericentralen Zonen beschränkt, sondern konnte auch für die periportalen Zonen demonstriert werden. 120 min nach der APAP Injektion konnte ein Influx an APAP-GSH und an Taurocholaten von den Gallengefäßen in die pericentrale Zone und von dort ins Blut beobachtet werden. Dieser massive Eintrag von Gallensäuren beweist, dass sich ein „sekundäres“ Toxizitätsevent ereignet, welches vormals durch die Aktivierung des APAPs in der Leber gestartet wurde.
Antibacterial secondary metabolites from four endophytic fungi, Eupenicillium sp., Diaporthe sp., Fusarium decemcellulare and Alternaria sp.
(2016) Li, Gang; Spiteller, Michael; Kayser, Oliver
Endophytes are microorganisms that colonize living, internal tissues of host plants for at least a part of their life cycle without causing any immediate, visible manifestation of disease. Endophytic microorganisms as a promising source of antibacterial natural products have attracted considerable attention. The main objective of this study was isolation, identification and antibacterial evaluation of secondary metabolites from endophytic fungi harbored in Chinese medicinal plants Xanthium sibiricum, Mahonia fortunei, and Lonicera japonica, which have been used in Traditional Chinese Medicine (TCM) for treating bacterial infection-related ailments. An endophyte, Eupenicillium sp. LG41 was isolated from the root of X. sibiricum. Twenty-two secondary metabolites including six decalin motif-containing compounds (five new) (1-6), two new sirenin derivatives (7 and 8), three pigments (one new) (9-11), eight paraconic acids and alkylitaconic acids (three new natural products) (12, 14, 16, 19, and 21-24), and three other known compounds (25-27), were isolated from the rice, PDB or modified PDB medium using the OSMAC approach. Four methylated derivatives (13, 15, 17, and 20) and an isomerization product (18) of paraconic acids were obtained for structure-activity relationship (SAR) analysis. The endophytic fungus Diaporthe sp. LG23 which was isolated from the leaves of M. fortunei collected from Shanghai, China, produced twelve metabolites including a novel tetracyclic triterpenoid with an aromatic B-ring system (28), seven biosynthetically related known steroids (29-35), together with four aromatic or glycosylated compounds (one new) (36-39). Further isolation and identification of endophytic fungi from M. fortunei (stem) collected from a different location, Guangdong, China, afforded an endophyte, Fusarium decemcellulare LG53. Chemical investigation led to the identification of eight compounds: three new cyclic pentapeptides (40-42), two cyclic lipopeptides (one new) (43 and 44), a new pyrone derivative (45), a known xanthone derivative (46), and a reported triterpenoid (47). Alternaria sp. LG19 was derived from the leaves of L. japonica. It was discovered to produce two aromatic metabolites (48 and 53), four well-known Alternaria mycotoxins including altenuene (49), isoaltenuene (50), alternariol (51) and altertoxin I (52), as well as a dicarboxylic acid (54). Altogether, forty-nine compounds were isolated from the above four endophytic fungi and five semi-synthetic derivatives were experimentally produced. Among the isolated compounds, fifteen compounds have new structures and three are new natural products which were reported as synthetic products in the literature. Their planar and relative structures were determined by the interpretation of spectroscopic data, such as IR, UV, HRMSn and NMR, and/or the single crystal X-ray diffraction study, and/or 13C NMR calculation. Their absolute configurations were deduced by one or more of following methods, ECD and optical rotation data, ECD and ORD calculations, Marfey’s method, and the single crystal X-ray diffraction study with a Cu-Kα radiation. The antibacterial efficacies of new compounds were investigated against several Gram-positive and Gram-negative bacteria obtained as clinical or environmental strains. For decalin-containing metabolites, eupenicinicol B (3) had the same efficacy as that of gentamicin against clinically relevant bacterium Staphylococcus aureus at the concentration of 1.0 µg/mL, while eupenicinicol D (5) was more active than 3 with an MIC value of 0.1 µg/mL. These data support the notion that altering the substitution at C-11 could drastically increase the inhibitory activity. The paraconic acid (2S,3R,4S)-4-methyl-5-oxo-2-pentyl-tetrahydro-furan-3-carboxylic acid (12) was discovered to only inhibit tested Acinetobacter spp., especially the multi-drug resistant strain Acinetobacter baumannii. The above data indicated a genus-specific antibacterial compound with a preferred stereochemical configuration. The novel tetracyclic triterpenoid 19-nor-lanosta-5(10),6,8,24-tetraene-1α,3β,12β,22S-tetraol (28) showed pronounced antibacterial efficacy against all the tested organisms, especially against the clinical isolates Streptococcus pyogenes and Pseudomonas aeruginosa. 5 and 28 also demonstrated marked cytotoxicity against human acute monocytic leukemia cell line (THP-1), while 12 exhibited no cytotoxic activity. The results reported in this thesis provide several antibacterial secondary metabolites worthy of further investigation, especially the paraconic acid (12, termed pacbactin), underlining the potential of endophytic fungi as a source of diverse antibiotics.
Crosstalk and antibacterial molecules from endophytes harbored in Narcissus tazetta and Buxus sinica
(2016) Wang, Wen-Xuan; Spiteller, Michael; Kayser, Oliver
Endophytic fungi and bacteria are microorganisms harbored in the plant tissues without causing any disease. They are proven to be a rich source of new anticancer and antimicrobial compounds as well as clinically valuable agents identified from plant materials. However, recent research encounters enormous difficulties, which reveals that endophytic systems are complex ecological communities maintained by inter-species and inter-kingdom interactions, including chemical crosstalk. Consequently, investigating the chemical basis of these interactions is essential to interpret the interaction among endophytes and host plant, and exploit the true potential of endophytes. Based on metabolite screening of endophytes from several Chinese traditional medicinal plants with antibacterial related uses, endophytic fungi and bacteria isolated from two plants Narcissus tazetta and Buxus sinica were selected for the investigation in this thesis. From an endophytic fungus, Fusarium solani N06, isolated from a bulb of N. tazetta, nine new hexacyclopeptides (1−9) were discovered as crosstalk molecules between it and another endophytic bacterium, Achromobacter xylosoxidans N12B, from the same tissue. In the matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) experiments, the secretion of these compounds from this F. solani N06 and the accumulation towards A. xylosoxidans N12B was visualized on the border between the fungus and bacterium. From the optimized culture of F. solani N06, sufficient extracts of these hexacyclopeptides were analyzed by liquid chromatography-multiple stage mass spectrometry (LC-MSn) and their sequence was identified to be cyclo((Hyp or Dhp)-Xle-Xle-(Ala or Val)-Thr-Xle) (Dhp: dehydroproline) based on the characteristic a, b, or y ions in MSn spectra. The phenomenon that coexisting endophytes utilize new signal molecules suggests that they have developed communication strategies to survive and function in their distinct ecological niches. Four new compounds, colletotrichones A−D (10−13) and one known compound chermesinone B (14a) were isolated from the fermented rice medium of an endophytic fungus Colletotrichum sp. BS4, which was isolated from the leaves of B sinica. With comprehensive spectroscopic methods including 1D and 2D NMR, HRMS, ECD spectra, UV, and IR as well as single crystal X-ray diffraction and quantum chemistry calculation, their structures were identified to be azaphilones sharing a 3,6a-dimethyl-9-(2-methylbutanoyl)-9H-furo[2,3-h]isochromene-6,8-dione core structure. In the antibacterial bioassay against two environmental strains of Escherichia coli and Bacillus subtilis, as well as two human pathogenic clinical strains Staphylococcus aureus and Pseudomonas aeruginosa, colletotrichone A (10) exhibited extraordinary activity against E. coli and B. subtilis, with a minimum inhibitory concentration lower than positive controls streptomycin and gentamicin. In the cytotoxic assay, interestingly, compounds 10−12, and 14a showed slight cytotoxicity at high concentration (100 µM). Furthermore, the spatial distribution and localization of the compounds produced by the endophyte were visualized by MALDI-imaging-HRMS, which revealed their plausible ecological functions. In the investigation of endophytic fungus Phyllosticta capitalensis harbored in the leaves of B. sinica, two new lactam-fused 4-pyrones (15, 16) were discovered in static PDB fermentation. Their structures were elucidated by 1D and 2D NMR, LC-MSn, DFT 13C NMR calculation and chemical reaction. By 16S rRNA analyses, an endosymbiotic bacterium Herbaspirillum sp. was discovered in the hyphae of P. capitalensis, which was not cultivable in ordinary culturing condition. The PKS/NRPS gene cluster analyses showed these two compounds were biosynthesized by fungal PKS and bacterial NRPS. This cross-species cooperation of secondary metabolites production unveiled another level of interaction between the endophytic bacterium and fungus.
Degenotoxification of pharmaceuticals by molecular imprinting and organic solvent nanofiltration
(2012-08-30) Székely, György; Sellergren, Börje; Weberskirch, Ralf
In dieser Arbeit wurden das molekulare Prägen (MI) und die Nanofiltration organischer Lösungsmittel (OSN) als Werkzeuge für die Entfernung genotoxischer Substanzen aus pharmazeutischen Stoffen untersucht. Die Herstellung von aktiven pharmazeutischen Wirkstoffen (APIs) beinhaltet oft die Verwendung von hochreaktiven Reagenzien welche als bedenkliche, genotoxische Reststoffe im Endprodukt verbleiben können. Genotoxische Verunreiningen (GTIs) stellen hierbei eine Gruppe von Stoffen dar, die elektrophile Reaktionen mit nucleophilen Gruppen der DNS eingehen können. In der pharmazeutischen Industrie ist das Interesse bezüglich der Entfernung von GTIs von höchster Bedeutung, da GTIs gegebnermassen ein erhöhtes Risiko für das pharmazeutische Produkt darstellen. Zwei herkömmliche API Aufreinigungstechniken, die Flash Chromatographie und die Umkristallisierung wurden in einer Machbarkeitsstudie mit OSN verglichen um deren Tragfähigkeit als Aufreiniungsschritt zu ermitteln. Hierbei wurden das Recycling des Lösungsmittels, Massenverluste, Kohlenstoffbelastung und Energieverbrauch berücksichtigt. Es wurde auch eine Kostenanalyse vorgnommen, um die wirtschaftlichen und umweltrelevanten Aspekte der verschiedenen Prozesse zu vergleichen. Dabei hat sich ergeben, dass OSN eine gleichwertige Leistung aufzeigt, weswegen die Untersuchung auf lösungsmittelresistente Membranen erweitert wurde. Eine Reihe von APIs und dazugehörigen GTIs wurden als Modelsubstanzen in verschiedenen Diafiltrationsversuchen untersucht. Dies erwies sich als technische Herausforderung, da GTIs ebenfalls von den Membranen ausgeschlossen wurden. Um die Entfernung von GTIs zu optimieren und die Verluste von API zu verringern, wurde der Einfluss von Druck auf den Ausschluss von gelösten Stoffen untersucht. In einer weiteren Studie wurden molekular geprägte Materialien auf ihre Selektivität bezüglich 1,3-Diisoproylurea (IPU) untersucht, da diese einen höheren Ausschluss aufweisen. Handelsübliche ExploraSep® Platten A basierend auf selektiv bindenden Harzen mit hydrophoben und Carbonsäure-Gruppen wurden auf Bindung zu IPU untersucht. Da diese jedoch eine unzureichende Funktionalität aufwiesen, wurden neue Harze mit Methacrylsäure und mit Hilfe eines Deprotonierungsreagenzes synthetisiert. Diese neuartigen geprägten Polymere und die entsprechenden nicht-geprägten Kontrollpolymere zeigten eine erfolgreiche Entfernung von GTIs von verschieden APIs. Der Vergleich von geprägten Polymeren und OSN ergab, dass die geprägten Harze besser bei geringen GTI Konzentrationen funktionieren und die OSN Membranen besser bei hohen. Diese Erkenntis führte dann zu einer synergistischen Kombination von OSN und MI um IPU zu entfernen und unspezifisch gebundenes API in einem selektiven Waschschritt vom Harz zu lösen. Mittels NMR wurde der Einfluss des Deprotonierungsreagenzes sowohl auf die Vorpolymerisation als auch auf die Wiedererkennungseigenschaften des Harzes untersucht. Es wurde beobachtet, dass sich Komplexe höherer Ordnung und auch Selbst-Assoziationen ausbilden, welche dann in der Bestimmung der Assoziationskonstanten berücksichtigt wurden. Die Einführung von Leistungs-Änderungs-Indikatoren, wie zum Beispiel Kapazitäts-Änderungs-Index, Beschleunigungs-Index und Gesamtleistungs-Indikatoren wurden vorgeschlagen.

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