Blood flukes of the genus Schistosoma are causative agents of the disease schistosomiasis, which affects more than 250 million people worldwide and together with malaria represents the most important parasitic infection. There is a high risk of resistance development against the only drug in use, therefore novel therapeutic approaches for schistosomiasis are intensively researched. Proteolytic enzymes of schistosomes are crucial for their survival in the host and thus are promising drug and vaccine targets. This thesis is focused on two proteases of the human blood fluke Schistosoma mansoni, which were produced as recombinant proteins and functionally characterized. The first one is serine protease SmSP2, which is localized at the surface of the adult worms and secreted into the blood of the host. It was identified as a vasodilatory and fibrinolytic agent, and its modulatory role in host-parasite interactions was proposed. The second one is cysteine cathepsin SmCL3, which is involved in the digestion of host blood proteins serving schistosomes as nutrients. Potent peptidomimetic inhibitors of SmCL3 were identified, and their antischistosomal activity was demonstrated in an assay with live parasites. The thesis provides new important information about S. mansoni proteases, their pathobiochemistry...

京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第19654号 / 薬博第824号 / 新制||薬||240(附属図書館) / 32690 / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 松原 和夫, 教授 中山 和久, 教授 金子 周司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13158号 / 論医博第2145号 / 新制||医||1029(附属図書館) / (主査)教授 川上 浩司, 教授 戸井 雅和, 教授 松田 秀一 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21699号 / 医科博第103号 / 新制||医科||7(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 妹尾 浩, 教授 渡邊 直樹, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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Most cancer cells rely on aerobic glycolysis to support the mitochondrial oxidative phosphorylation system (OXPHOS). The persistent oxic-anoxic cycle exerts selection pressures which lead to constitutive activation of glycolysis even in the presence of abundant oxygen. Expression of hypoxia-inducible factor 1 (HIF1) increases following hypoxia in neoplastic cells. This leads to the induction of pyruvate dehydrogenase kinase 1 (PDK1). The latter inactivates pyruvate dehydrogenase (PDH) that converts pyruvate to acetyl-coenzyme A for delivery to the tricarboxylic acid cycle (TAC). Dichloroacetic acid (DCA) is an inhibitor of PDK that forces cells into oxidative phosphorylation thereby suppressing cancer growth. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (C9), along with a few other 17β-estradiol analogs, are a novel class of in silico-designed inhibitors of microtubule dynamics. These newly designed and synthesized antimitotic compounds induce G2/M arrest and apoptosis by docking to colchicine binding site between α- and β-tubulin. These compounds are 5 to 20 times more potent than their source molecule, 2-methoxyestradiol (2ME). To improve bioavailability C9 has been in silico-modified at carbon positions C2, C3 and C17 compared to 2ME. The approach to investigate the anticancer potential of the in silico-designed antimitotic C9 in combination with the glycolytic inhibitor DCA in vitro is novel. Human breast carcinoma cell line MCF-7 and non-tumorigenic breast cells MCF-12A were used as an experimental model system. The present study demonstrated that DCA (7.5 mM) in combination with C9 (130 nM) selectively inhibited half of MCF-7 cells‘ population (50.8%). Under the same treatment conditions, MCF-12A cells displayed high number of cell survival (70% cell growth). Qualitative morphological studies revealed decreased cell density in both cell lines, as well as hallmarks of apoptosis and autophagic processes including formation of apoptotic bodies, DNA fragmentation and autophagic vacuoles. Cell cycle- and apoptosis quantification analyses revealed C9+DCA treatment induced apoptosis in both cell lines and exhibited selectivity towards tumorigenic cells. Presence of autophagosome was observed and microtubule-associated protein 1 light chain 3 (II) (LC3-II) expression was elevated. Reduction of mitochondrial membrane potential depolarization in tumorigenic MCF-7 cells was demonstrated, but not in MCF-12A cells. Oxidative stress tests suggested the combination treatment C9+DCA is able to induce lysosomal rupture and/or mitochondrial damage in tumorigenic MCF-7 cells. Kinase inhibition studies revealed that transient activation of c-Jun N-terminal kinase (JNK) plays an important role in cell proliferation. However, C9+DCA stimulated prolonged JNK activation and, in turn, promoted Bcl-2 phosphorylation, thereby facilitating autophagic and apoptotic cell death. C9+DCA induced expression of a number of genes related to stress in MCF-7 treated cells including TP53BP1, MDM2 and BBC3/PUMA. Genes related to cell motility and maintenance of the cytoskeleton such as ACTG1, MAP7, TUBA1, TUBA6, TUBA8 and TUBB2A genes were down-regulated. In MCF-12A cells, treatment of C9+DCA induced expression of multidrug resistance gene ABCB1. Moreover, genes involved in reactive oxygen species metabolism FTH1, GSTA2, NOS2A, SMOX, SOD1 and SOD2 were also up-regulated. In conclusion, the novel 17β-estradiol derivative, C9, in combination with DCA is a potent antiproliferative treatment. This study addressed the mechanisms of combination treatment at the basis of molecular and cellular level, warranting further research projects to develop viable and functional combination treatment as clinically useable anticancer agents. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Physiology / PhD / Unrestricted

SRY (sex determining region Y)-box 2 (SOX2) is one the embryonic stem cell transcription factors that is capable of reprogramming adult differentiated cells into an induced pluripotent cell. SOX2 is amplified in various types of epithelial cancers and its high its expression correlates with poor prognosis and decreased patient survival. Aberrant Wnt signaling drives the colo-rectal carcinogenic process and is a major determinant of the disease outcome. This study demonstrates that SOX2 counteracts Wnt driven tumor cell proliferation and maintains quiescence in a sub-population of Colo-Rectal Cancer (CRC) cells. High SOX2 expression is found in a sub-group of CRC patients with advanced disease. High SOX2 expression coupled with low Wnt activity was found in SW620 metastatic CRC cell line, while the opposite was true for the isogenic SW480 primary tumor cell line. SOX2 silencing increased Wnt activity and enhanced the oncogenic potential of SW620 cells in vitro and in vivo while over-expression had opposite effects in SW480 cells. SOX2 up-regulates the expression of PTPRK and PHLPP2 protein phosphatase genes which in turn attenuates Wnt activity by interfering with Protein Kinase A, B and C mediated beta catenin phosphorylation at Serine 552 and 675 amino acid residues thereby diminishing its nuclear sequestration and transcriptional activation. Thus SOX2 mitigates growth factor mediated Wnt activation in CRC cells and inhibits cellular proliferation so that these cells are forced to change their oncogene addiction. In effect, high SOX2 expression causes clonal evolution of APC mutant CRC cells from a state of high Wnt dependency to a state of low Wnt dependency in the process making such cells resistant to Wnt inhibitor therapy. Enhanced SOX2 transcriptional activity was associated with increased proportion of cancer cells in G0-G1 phase of cell cycle. Changing SOX2 protein levels in cells had a direct correlation with mRNA levels of RBL2-HUMAN and CDKN2B genes, which serve as regulators of G0 and G1 respectively. SOX2 was shown to physically bind and to the promoter region of these two genes and enhance their transcription. Thus high SOX2 expression, up-regulates the expression of key cell cycle inhibitor genes like RBL2 and CDKN2B and keeps cells in a dormant state. This phenomenon allows colon cancer cells to escape from cytotoxic drug therapy directed at rapidly dividing cells and cause treatment failure and disease relapse.

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The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases. For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R. This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist. For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability. The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab. / Die Zytokine Interleukin-4 (IL-4) und IL-13 sind zentrale Mediatoren in der humoralen Immunantwort und sind wesentlich an der Entstehung chronisch inflammatorischer Erkrankungen, wie Asthma, Allergien und atopischer Dermatitis beteiligt. Daher werden IL- 4 und IL-13 als wichtige therapeutische Angriffspunkte für die Behandlung atopischer Erkrankungen betrachtet. Zur Signaltransduktion aktiviert IL-4 zwei heterodimere Rezeptorkomplexe, den Typ I Rezeptor bestehend aus den Untereinheiten IL-4R und γc und den Typ II Rezeptor, der sich aus IL-4R und IL-13R1 zusammensetzt. Der Typ II Rezeptor wird auch von IL-13 zur Signalweiterleitung genutzt, wodurch sich die teilweise überschneidenden Funktionen der beiden Zytokine erklären lassen. Die überlappende Rezeptornutzung erlaubt es durch eine gezielte Blockade des IL-4R-Rezeptors sowohl IL-4, als auch IL-13 gleichzeitig zu inhibieren. Ziel dieser Arbeit war die Herstellung neuartiger IL-4 basierter Antagonisten. Dazu wurden ausgewählte Positionen innerhalb der IL-4 Bindestelle für γc and IL-13R1, jedoch außerhalb des Bindeepitops für IL-4R gezielt chemisch modifiziert. Im Gegensatz zu bereits existierenden Studien, die synthetische Gruppen wie Polyethylenglykol (PEG) zur chemischen Modifizierung von IL-4 nutzten, wurden in dieser Arbeit Glykane als natürliche Alternative zu PEG an IL-4 gekoppelt. Da sich Glykosylierung auf wichtige pharmakologische Eigenschaften proteinbasierter Therapeutika, wie Immunogenität oder Serum Halbwertszeit, positiv auswirken kann, würde der Zucker in dieser Strategie nicht nur den auf sterischer Hinderung basierenden inhibitorischen Effekt vermitteln, sondern könnte gleichzeitig zu einer Optimierung der pharmakologischen Eigenschaften des IL-4 Inhibitors beitragen. Zur chemischen Zucker-Kopplung wurden zusätzliche Cystein-Reste in IL-4 eingebracht, deren freie Thiol-gruppen eine hochspezifische Modifizierung der IL-4 Mutanten erlauben. Die IL-4 Cystein-Mutanten wurden rekombinant in prokaryotischen und eukaryotischen Expressionssystemen hergestellt. Anschließend wurden verschiedene Strategien entwickelt, die eine ortsspezifische Kopplung Amin- und Thiol-haltiger Zucker an die eingebrachten Cystein-Reste in IL-4 ermöglichen. Eine Linker-basierte Reaktion, sowie ein Kopplungsansatz, der eine Aktivierung der Thiol-Gruppe mittels Bromselenobenzol erforderte, wiesen einige Nachteile auf, die eine erhebliche Einschränkung ihrer technischen Durchführbarkeit zur Folge hatte. Eine dritte Strategie hingegen, die eine Rückfaltung derIL-4 Cystein-Mutanten in Anwesenheit von Thiol-Glykanen involvierte, erlaubte eine effiziente Herstellung von IL-4 Glykokonjugaten in Form gemischter Disulfide im Milligrammbereich. Dieser Ansatz könnte somit zur Produktion homogen glykosylierter IL-4 Antagonisten im Großmaßstab eingesetzt werden. Die Herstellung homogener Glykokonjugate mit klar definierten Glykosylierungsmuster würde es erlauben über die gekoppelten Glykanstrukturen die pharmakologischen Eigenschaften von IL-4, wie Absorption und metabolische Stabilität, gezielt zu modulieren. Die hergestellten IL-4 Glykokonjugate erwiesen sich als hochwirksame Antagonisten, die die Aktivität von IL-4 und IL-13 in zellbasierten Experimenten inhibieren konnten. Glykosylierte IL-4 Antagonisten stellen somit eine vergleichbare Alternative zu gängigen IL-4 Inhibitoren dar, die zur Behandlung von atopischen Erkrankungen eingesetzt werden, wie beispielweise der neutralisierende anti-IL-4R Antikörper Dupilumab.