Universitätsbibliothek

Die verfügbaren in vitro Genotoxizitätstests weisen hinsichtlich ihrer Spezifität und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschränkungen auf. Um diese Mängel zu überwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Prüfung auf Genotoxizität in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizitätsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbrüche), phosphorylierten H3 (S10) (mitotische Zellen), nukleären Protein p53 (Genotoxizität) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und ergänzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre Fähigkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen“ gegenüber der „alten“ Methode führte zu einer verbesserten Sensitivität von 95% gegenüber 90%, Spezifität von 90% gegenüber 72% und einer MoA-Klassifizierungsrate von 85% gegenüber 45% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgewählter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen können. Die Kombination der höchstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 ermöglichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivität, Spezifität und Prädiktivität von 86%, 83% und 85%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizität mit TP53I3, Klastogenität mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen für BubR1 (S670) und pH3 (S28) mit Aneugenität, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenität, p53 (K373) mit Genotoxizität und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinveränderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivitäten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden können und daher eine bessere Risikobewertung von Wirkstoffkandidaten ermöglichen.

For mutagenicity testing, primary lymphocytes or mammalian cell lines are employed. However, the true target for carcinogenic action of mutagenic chemicals may be stem cells. Since hematopoietic cancers induced by chemical agents originate at the hematopoietic stem cell (HSC) stage and since one of the side effects of chemotherapeutic cancer treatment is the induction of secondary tumors, often leukemias, HSC may be a suitable cell system. We compared the sensitivity of HSC with the genotoxicity testing cell line TK6 for chromosomal mutations. HSC were less sensitive than TK6 cells for the genotoxic effects of the model genotoxins and chemotherapeutic agents doxorubicin, vinblastine, methyl methanesulfonate (MMS) and equally sensitive for mitomycin C (MMC). However, loss of viability after mitomycin C treatment was higher in HSC than in TK6 cells. Among the factors that may influence sensitivity for genomic damage, the generation or response to reactive oxygen species (ROS) and the effectiveness of DNA damage response can be discussed. Here we show that HSC can be used in a standard micronucleus test protocol for chromosomal mutations and that their sensitivity was not higher than that of a classical testing cell line.

Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer. Still, most of the mammalian HAD phosphatases remain functionally uncharacterized. This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos. To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation. The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells. These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling. Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase. To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions. Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under hypoxic (~1% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3’-phosphate (GADP). Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism. Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.

G protein-coupled receptors (GPCRs) form the biggest receptor family that is encoded in the human genome and represent the most druggable target structure for modern therapeutics respectively future drug development. Belonging to aminergic class A GPCRs muscarinic Acetylcholine receptors (mAChRs) are already now of clinical relevance and are also seen as promising future drug targets for treating neurodegenerative diseases like Alzheimer or Parkinson. The mAChR family consist of five subtypes showing high sequence identity for the endogenous ligand binding region and thus it is challenging until now to selectively activate a single receptor subtype. A well accepted method to study ligand binding, dynamic receptor activation and downstream signaling is the fluorescence resonance energy transfer (FRET) application. Here, there relative distance between two fluorophores in close proximity (<10 nm) can be monitored in a dynamic manner. The perquisite for that is the spectral overlap of the emission spectrum of the first fluorophore with the excitation spectrum of the second fluorophore. By inserting two fluorophores into the molecular receptor structure receptor FRET sensors can serve as a powerful tool to study dynamic receptor pharmacology. Dualsteric Ligands consist of two different pharmacophoric entities and are regarded as a promising ligand design for future drug development. The orthosteric part interacts with high affinity with the endogenous ligand binding region whereas the allosteric part binds to a different receptor region mostly located in the extracellular vestibule. Both moieties are covalently linked. Dualsteric ligands exhibit a dynamic ligand binding. The dualsteric binding position is characterized by a simultaneous binding of the orthosteric and allosteric moiety to the receptor and thus by receptor activation. In the purely allosteric binding position no receptor activation can be monitored. In the present work the first receptor FRET sensor for the muscarinic subtype 1 (M1) was generated and characterized. The M1-I3N-CFP sensor showed an unaltered physiological behavior as well as ligand and concentration dependent responses. The sensor was used to characterize different sets of dualsteric ligands concerning their pharmacological properties like receptor activation. It was shown that the hybrids consisting of the synthetic full agonist iperoxo and the positive allosteric modulator of BQCA type is very promising. Furthermore, it was shown for orthosteric as well as dualsteric ligands that the degree of receptor activation is highly dependent on the length of and the chemical properties of the linker moiety. For dualsteric ligands a bell-shaped activation characteristic was reported for the first time, suggesting that there is an optimal linker length for dualsteric ligands. The gained knowledge about hybrid design was then used to generate and characterize the first photo-switchable dualsteric ligand. The resulting hybrids were characterized with the M1-I3N-CFP sensor and were described as photo-inactivatable and dimmable. In addition to the ligand characterization the ligand application methodology was further developed and improved. Thus, a fragment-based screening approach for dualsteric ligands was reported in this study for the first time. With this approach it is possible to investigate dualsteric ligands in greater detail by applying either single ligand fragments alone or in a mixture of building blocks. These studies revealed the insights that the effect of dualsteric ligands on a GPCR can be rebuild by applying the single building blocks simultaneously. The fragment-based screening provides high potential for the molecular understanding of dualsteric ligands and for future screening approaches. Next, a further development of the standard procedure for measuring FRET by sensitized emission was performed. Under normal conditions single cell FRET is measured on glass coverslips. After coating the coverslips surface with a 20 nm thick gold layer an increased FRET efficiency up to 60 % could be reported. This finding was validated in different approaches und in different configurations. This FRET enhancement by plasmonic surfaces was until yet unreported in the literature for physiological systems and make FRET for future projects even more powerful.

In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e. g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.