Brain metastases (BMs) present a formidable obstacle across various primary cancer types, notably small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), melanomas, and breast cancers. In this investigation, we aim to evaluate the potential of genotype-guided targeted therapy while addressing the challenges of co-existing genomic alterations frequently encountered in BMs. This research explores the efficacy of adagrasib (MRTX849), a KRAS G12C inhibitor, and abemaciclib, a CDK 4/6 inhibitor, both individually and in combination against BMs originating from NSCLC cell lines harboring KRAS G12C and CDKN2A mutations. Utilizing a diverse array of methodologies encompassing cell viability assays, cell death assays, western blot analyses, and in vivo xenograft models, we elucidate both the therapeutic potential and underlying mechanisms. Distinct responses to adagrasib and abemaciclib monotherapies were observed across two different cell lines, underscoring the necessity for tailored treatment strategies. While adagrasib exhibited variable efficacy, abemaciclib consistently inhibited CDK 4/6 activity. Notably, the combination therapy demonstrated synergistic effects, suggesting a promising approach for enhanced therapeutic outcomes. Our findings from both in vitro assays and western blot analyses corroborate targeted pathway inhibition, although the observed pathway reactivation underscores the importance of optimizing dosing strategies. In vivo studies further support our in vitro findings, demonstrating efficacy but also raising concerns regarding toxicity with combination therapy. Pharmacokinetic / pharmacodynamic (PK/PD) analyses underscore potential advantages of combination therapy in terms of systemic exposure and brain penetration. Despite histological evidence of therapeutic effects, discrepancies between in vivo and in vitro caspase-dependent apoptosis results highlight the complexity of tumor biology and the challenges of translation. By Focusing on personalized treatment approaches and addressing therapeutic hurdles, this work establishes the foundation for clinical investigation in advancing the management of BMs and improving treatment outcomes in NSCLC patients.

Malaria, a disease caused by members of the Plasmodium genus, remains a threat to global health. Despite the availability of therapeutics, Plasmodium's propensity for generating resistance-conferring mutations threatens the efficacy of these drugs. Therefore, it is essential to develop novel therapeutics, and one approach to discover such compounds is to repurpose current drugs as antimalarials. Human kinase inhibitors, most of which are developed as antineoplastics, are a valuable source of such novel compounds. Human kinase inhibitor research spans over twenty years, generating a wellspring of knowledge regarding compound design, mechanism, and tolerability that can be leveraged in the quest to develop new antiplasmodial drugs. Furthermore, the plasmodial kinome differs substantially from the human kinome, providing opportunities for selectivity and minimization of off-target effects in the host. To this end, we sought to identify and characterize compounds within human kinase inhibitor collections that have antiplasmodial effects. One library yielded a potent polo-like kinase 1 (PLK1) kinase inhibitor, BI-2536, which possessed potent antiplasmodial activity in both the asexual blood stage and liver stage and likely acts through involvement of amino acid starvation. Another library comprised exclusively of type II kinase inhibitors, designed to target kinases in the inactive conformation, produced several interesting lead compounds – TL5-135, YLIU-06-026-1, and the analog pair XMD13-99 and WZ9-034-2. These compounds were highly active against asexual blood stage parasites, killing rapidly while also possessing favorable selectivity and liver stage activity. In vivo, TL5-135 and YLIU-06-026-1 acted prophylactically by preventing infection, and therapeutically by resolving an established infection. Currently, investigations are underway to determine the mechanism of action of the lead compounds and to improve their druglike properties. In whole, this effort has not only yielded promising antiplasmodial compounds, but it also underscores the value of the repurposing approach in the quest for novel antimalarial drugs.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25487号 / 医博第5087号 / 新制||医||1073(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小濱 和貴, 教授 妹尾 浩, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...

Filarien persistieren bis zu 15 Jahren in ihren Wirten. Als eine Ursache dieser Persistenz diskutiert man die Fähigkeit der Filarien, die Immunantwort des Wirtes gezielt zu modulieren und eine zelluläre Hyporeaktivität zu induzieren. In der vorliegenden Arbeit wurde untersucht, ob ein sezernierter Cysteinproteasen-Inhibitor (Onchocystatin) der humanpathogenen Filarie Onchocerca volvulus immunmodulierende Eigenschaften besitzt und an der Herausbildung eines hyporeaktiven Immunstatus des Wirtes beteiligt ist. Für die Untersuchungen wurde Onchocystatin als full length Molekül (rOv17) und als verkürztes Molekül (trOv17) rekombinant hergestellt. Das verkürzte trOv17 besitzt aufgrund des Fehlens des N-terminalen Bereiches eine verminderte proteaseinhibitorische Aktivität. Die in vitro Studien mit den rekombinant hergestellten O. volvulus Cystatinen verdeutlichen, daß rOv17 und auch trOv17 potente Immunmodulatoren sind, die sowohl die antigenspezifische als auch die polyklonal-stimulierte Proliferation von humanen PBMC inhibieren. Die zelluläre Hyporeaktivität ist dabei auf die Modulation von Monozytenfunktionen zurückzuführen. rOv17 und trOv17 modulieren die Antigenpräsentation, die Zytokinproduktion und die Expression kostimulatorischer Signale von humanen Monozyten. So konnte gezeigt werden, daß rOv17 und trOv17 die Aktivität von humanem Cathepsin L und S inhibieren und die Expression von HLA-DR, CD40 und CD86 vermindern. Die Modulation der Zytokinproduktion durch rOv17 und trOv17 ist durch eine verstärkte TNF-alpha und IL-10 Produktion und durch eine verminderte IL-12 Produktion charakterisiert. Desweiteren konnte in Neutralisationsstudien mit anti-IL-10 Ak gezeigt werden, daß die verminderte Expression von HLA-DR, CD40 und CD86 Folge der durch rOv17 und trOv17 induzierten verstärkten IL-10 Produktion ist. Im Gegensatz dazu bleibt die verminderte IL-12 Produktion und die verminderte polyklonal-stimulierte Proliferation humaner PBMC auch nach der Neutralisation von IL-10 bestehen. Die Studien mit den rekombinant hergestellten O. volvulus Cystatinen zeigen, daß rOv17 und trOv17 ihr immunologisches Umfeld auf vielfältige Art und Weise modulieren. Dabei spielt vermutlich die Inhibition der Aktivität einer Wirtscysteinprotease, aber auch ein von der Funktion als Cysteinproteasen-Inhibitor unabhängiger Mechanismus eine Rolle. Der Cysteinproteasen-Inhibitor der Filarie O. volvulus besitzt somit die Eigenschaft, die Immunantwort des Wirtes zu modifizieren, und ist vermutlich eine wesentliche Komponente, die dem Parasiten eine lange Persistenz im Wirt ermöglicht. / Immune responses of individuals infected with filarial nematodes are characterized by a marked cellular hyporesponsiveness. The establishment of this hyporesponsiveness is considered as an important mechanism to avoid host immune responses which could eliminate the parasites. The present study is investigating the immunomodulatory potential of a 17 kD secreted cysteine protease inhibitor (onchocystatin) of the human pathogenic filaria Onchocerca volvulus. In vitro studies using recombinant onchocystatin (rOv17) identified this inhibitor as a potent immunomodulator. rOv17 suppresses the antigen-driven and the polyclonally-stimulated proliferation of human PBMC. This cellular hyporeactivity is due to the modulation of monocytic function by rOv17, comprising the modulation of antigenpresentation, the expression of costimulatory molecules and the production of cytokines. Thus rOv17 strongly inhibits the activity of human cathepsin L and S and reduces the expression of MHC class II molecules as well as the expression of CD40 and CD86 on human monocytes. The modulation of cytokine production by rOv17 is characterized by an initial increase of TNF-alpha which is followed by an increase of IL-10 and a decrease of IL-12. By neutralization studies it was shown that the suppression of MHC class II molecules and of CD86 and CD40 is mediated by the rOv17 induced increase of IL-10. In contrast cellular hyporeactivity and the reduced IL-12 production remain unaffected by neutralization of IL-10. In comparison to rOv17 a truncated onchocystatin (trOv17) with lowered protease inhibitory activity was investigated. Surprisingly even trOv17 is immunomodulatory active suggesting that immunomodulation by onchocystatin is mediated by both an inhibitor-dependent and an inhibitor-independent mechanism. These data demonstrate that onchocystatin is a potent immunomodulator of host immune responses and in consequence is an essential component that enables the parasites a long persistence within their hosts.

S100A4 spielt eine zentrale Rolle für die Metastasierung des Dickdarmkrebses. Die Hemmung der S100A4 Expression stellt damit einen vielversprechenden therapeutischen Ansatz dar. Die vorliegende Arbeit präsentiert Niklosamid und Calcimycin als neue Inhibitoren der S100A4 Transkription. In Kolonkarzinomzellen, die mit einem der beiden Inhibitoren behandelt wurden, wurde die S100A4 Expression konzentrations- und zeitabhängig unterdrückt. Des Weiteren war die Zellmigration und -invasion in Abhängigkeit von S100A4 in behandelten Zellen vermindert. Niklosamid und Calcimycin Behandlung verhinderten die Zellproliferation und die Koloniebildung von Kolonkarzinomzellen. Beide Inhibitoren hemmten den konstitutiv aktiven Wnt Pathway von Kolonkarzinomzellen. Calcimycin Behandlung verminderte die Expression von beta-catenin. Niklosamid hemmte die Bildung des beta-catenin/TCF Komplexes und unterband damit die Expression von Wnt Pathway Genen, wie z.B. S100A4. Im Rahmen dieser Arbeit wurde ein in vivo Tiermodell entwickelt, mit dem die S100A4-induzierte Metastasierung mit Hilfe von nicht-invasivem Biolumineszenz Imaging visualisiert werden konnte. In diesem Model konnte gezeigt werden, dass Niklosamid signifikant die S100A4 Expression im Tumor vermindert und damit die Metastasierung hemmt. Des Weiteren zeigt diese Arbeit, dass S100A4 die Expression des Wnt Pathway Antagonisten DKK-1 in Kolonkarzinomzellen hemmt. DKK-1 selbst konnte als endogener Inhibitor der S100A4 Expression identifiziert werden. Zusammenfassend beschreibt die vorliegende Arbeit einen neuen regulativen Mechanismus im Wnt Pathway, der die S100A4 Expression im Kolonkarzinom fördert. Diese Beobachtung verdeutlicht die Notwendigkeit für wirksame S100A4 Inhibitoren, wie Niklosamid und Calcimycin, die das Potenzial haben, in einer klinischen Anwendung die Metastasierung von Kolonkarzinompatienten mit erhöhter S100A4 Expression zu hemmen und damit deren Überlebenschance wesentlich zu verbessern. / S100A4 promotes metastasis in colon cancer patients thereby reducing their five-year survival chances to less than 10%. Consequently, inhibition of S100A4 expression is a promising strategy for anti-metastatic treatment of colon cancer patients. The present study characterizes the small molecules niclosamide and calcimycin as transcriptional inhibitors of S100A4 which reduced S100A4 expression concentration- and time-dependently. Niclosamide and calcimycin treatment restricted cell migration, invasion and wound healing capabilities in a S100A4-specific manner, and inhibited cell proliferation and colony formation of colon cancer cells. Both small molecule inhibitors interfere with the constitutively active Wnt pathway. Targeting β-catenin expression by calcimycin or interfering with the β-catenin/TCF transcription activating complex by niclosamide resulted in reduced Wnt target gene transcription, among them S100A4. The study further presents a human colon cancer xenograft mouse model for monitoring S100A4-induced metastasis formation via non-invasive bioluminescence imaging. Treatment of xenograft mice with niclosamide resulted in a significant reduction of the S100A4 mRNA level in the tumor accompanied by inhibition of metastasis formation. Moreover, this study presents evidence that S100A4 is an inhibitor of DKK-1 expression. In colon cancer cells DKK-1 and S100A4 expression was negatively correlated. Ectopic S100A4 overexpression inhibited DKK-1 expression. Targeting S100A4 via shRNA recovered the repressed DKK-1 expression and vice versa. In summary, the study describes a novel positive feedback loop in the Wnt pathway regulation formed by S100A4 repressing its antagonist DKK-1. This novel mechanism further strengthens the need for S100A4 inhibitors such as niclosamide or calcimycin. Consequently, such small molecules provide immense potential for the treatment of colon cancer patients who are at high risk for S100A4-induced colon cancer metastasis.

In dieser Dissertation versuche ich, die zentralen Nitrogen-heterocyclischen Kerne, die Seitenketten und deren Position zu variieren. Als synthetische Strategie wurden Palladium-katalysierte Kupplungsreaktionen verwendet, um Seitenketten und Aryl-Substituenten einzuführen. Halogensubstituierte Diarylheterocyclen sind wichtige Intermediate in der Synthese der allgemeine Strukture. Die Einführung der gewünschten Seitenketten durch Carbon-Carbon und Carbon-Nitrogen-Bindungsknüpfung wurde durch Sonogashira-Kupplung, Heck-Kupplung und Buchwald-Hartwig-Aminierung erzielt. Mit der Sonogashira-Reaktion kann eine funktionalisierte Alkynylgruppe in die heterocyclischen Kerne effektiv und bequem eingeführt werden. Eine anschliessende katalytische Hydrierung der Alkynylgruppe führt zu funktionalisierten Alkyl substituierten Diarylheterocyclen. In der vorliegenden Arbeit wurden mehr als 180 Substanzen synthetisiert. Unter ihnen sind ungefähr 130 neue Substanzen. 86 von ihnen passen in die allgemeine Strukture. / In the present thesis, I tried to vary the central nitrogen-heterocyclic cores, the functionalised side chains and its position of attachment. As a synthetic strategy, palladium-catalyzed coupling reactions were used to introduce side chains and aryl substituents into the central heterocycle. In this way the utility of such reactions to heterocyclic systems, which were neglected so far, could be figured out. Halogen substituted diaryl heterocycles are important intermediates in the synthesis of general structures. The introduction of the desired side chains by Carbon-Carbon bond formation reactions was achieved by Sonogashira coupling and Heck coupling. Buchwald-Hartwig amination and nucleophilic substitution were used to establish side chains which are connected to the core heterocycle by heteroatom-Carbon bonds. Sonogashira reaction turned out to be the most effective and convenient method to introduce functionalized alkynyl group into the heterocyclic cores. In the present work, more than 180 compounds were synthesized. Among them, about 130 compounds are new products. 86 of them fit into the general structure.

Les pathologies vasculaires placentaires sont fréquentes et graves. La forme maternelle prédominante est la pré-éclampsie et la forme fœtale le retard de croissance intra-utérin. Les questions posées autour de ce sujet concernent tout d'abord la prédiction de la survenue de ces pathologies suffisamment tôt afin de permettre une surveillance rapprochée, une administration de corticoïdes et une prise en charge dans une maternité de niveau adaptée. La prévention de la survenue et de la récidive ainsi que le traitement de ces pathologies la phase constituée sont aussi des problématiques encore non résolues. Notre objectif était de travailler sur ces différentes questions pa l'intermédiaire de trois études : l'étude ANGIOPRED), l'étude VOLUPLA et l'étude GROWTH. Les résultats de ces travaux et une revue d la littérature mettent en évidence une perturbation des facteurs de l'hémostase et des facteurs angiogéniques dans la pré-éclampsie et dans le retard de croissance. L'association des facteurs maternels, échographiques, angiogéniques et sériques constitue un modèle prédictif efficace principalement du fait d'une excellente valeur prédictive négative. Le volume placentaire est corrélé au taux de D- Dimères et est intéressant pour la prédiction des pathologies vasculaires placentaires. De nouveaux travaux devront poursuivre l'étude d la prédiction, de la prévention et du traitement des pathologies liées au placenta. Le traitement est notamment l'objet de l'étude Growth qui vise à évaluer l'efficacité de l'énoxaparine dans le traitement du retard de croissance vasculaire constitué. / Placenta-mediated adverse pregnancy outcomes are frequent and severe pathologies whose predominant maternal form is preeclampsia and fetal form, intrauterine growth retardation. The questions asked about this subject concern first of all the prediction of the occurrence of its pathologies in a sufficiently early way to allow for close monitoring, administration of corticosteroids, and management in an appropriate level of maternity. The prevention of the occurrence and recurrence and the treatment of its pathologies in the constituted phase are also unresolved problems. Our objective was therefore to work on its various questions through three studies: the ANGIOPREI study, the VOLUPLA study and the GROWTH study. The results of his work and of the literature show that the factors of haemostasis anc angiogenic factors are disturbed in preeclampsia and in growth retardation. The association of maternal, ultrasound, angiogenic and serum factors constitutes a predictive model that is effective mainly by an excellent negative predictive value. The placental volume is correlated with the D-dimer level and is interesting for placenta-mediated adverse pregnancy outcomes prediction. New studies will have to continue the exploration of the prediction, prevention and treatment of this pathologies related to the placenta. The treatment is notably the object of the study Growth which aims to evaluate the effectiveness of the Enoxaparin for the treatment of constituted vascular growt retardation.

Introdução: As proteinases tem um papel importante no desenvolvimento, na destruição tecidual e na produção de muco causada pela DPOC. O inibidor de proteinase de origem vegetal Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) inibe tanto as proteinases da classe serina quanto da classe cisteína. Objetivos: Avaliar os efeitos do tratamento com EcTI nas alterações pulmonares induzidas pela elastase em camundongos. Métodos: Camundongos C57Bl6 receberam elastase via intratraqueal (50 uL/animal, grupo ELA) ou salina (grupo SAL). Os camundongos foram tratados com EcTI (2mg/kg) nos dias 1, 15 e 21 após a instilação de elastase (grupo ELA-EcTI) ou salina (grupo SAL-EcTI). No dia 28 do protocolo, os animais foram anestesiados, a mecânica pulmonar foi medida e o óxido nítrico exalado coletado. Posteriormente, foi realizado o lavado broncoalveolar e os pulmões foram removidos para a preparação de lâminas de histoquímica e imunohistoquímica. Por meio de morfometria analisamos o número de células positivas para neutrófilos, TNF-alfa, MMP-9, MMP-12, TIMP-1, iNOS, eNOS assim como a fração de volume de 8-iso-PGF2alfa, fibras colágenas e elásticas, nos septos alveolares e nas vias aéreas. Também foram avaliados o número de células positivas para macrófagos nos septos alveolares e MUC5ac nas vias aéreas. Resultados: O inibidor de proteinase EcTI reduziu as alterações de mecânica pulmonar (Ers, Htis e Raw), destruição do septo alveolar (Lm) e o número de células no lavado broncoalveolar (células totais, macrófagos, neutrófilos, linfócitos e eosinógilos) induzidos pela elastase. Em relação a resposta inflamatória, o EcTI reduziu o número de neutrófilos e de células TNFalfa positivas no septo alveolar e nas vias aéreas além de reduzir o número de macrófagos no septo alveolar. Considerando o remodelamento de matriz extracelular, o inibidor de proteinase atenuou a fração de volume de fibras colágenas e o número de células MMP-9 e MMP-12 positivas nos septos alveolares e nas vias aéreas. Além disso, nas vias aéreas ocorreu uma atenuação da fração de volume de fibras elásticas, e nos septos alveolares uma atenuação da quantidade de células que expressam TIMP-1. Em relação a resposta de estresse oxidativo, o EcTI reduziu a fração de volume de isoprostano e o número de células iNOS e eNOS positivas tanto nos septos alveolares quanto nas vias aéreas. O EcTI também reduziu o número de células MUC5ac positivas nas vias aéreas. Conclusões: O tratamento como inibidor EcTI modulou a mecânica pulmonar e reduziu as alterações inflamatórias, de remodelamento e de estresse oxidativo induzidas pela elastase intratraqueal. Embora sejam necessários mais estudos para elucidar os mecanismos envolvidos neste processo, o inibidor de proteinase EcTI pode ser considerado como um potencial instrumento terapêutico para o tratamento da DPOC / Background: Proteinases play a key role on emphysema development, tissue destruction and mucus production. Enterolobium contortisiliquum Tripsin Inhibitor (EcTI) is a proteinase inhibitor from plant that neutralizes serine and cysteine proteinases. Aims: To evaluated the effects of the EcTI treatment in pulmonary alterations induced by elastase in mice. Methods: C57Bl6 mice received elastase intratracheally (50 uL/animal, ELA group) or saline (SAL group). Afterwards, mice were treated with EcTI (2 mg/kg) at days 1, 15 and 21 after elastase instillation (ELA-EcTI group). Control group received saline and EcTI using the same protocol (SAL-EcTI group). At day 28, mice were anesthetized, respiratory mechanics were collected, and exhaled nitric oxide were analyzed. Afterwards, broncoalveolar lavage fluid was obtained and lungs were removed to perform histochemistry and immunohistochemistry stains. By morphometry, the number of neutrophils, TNF-alfa, MMP-9, MMP-12, TIMP-1, iNOS, eNOS positive cells as well as the volume proportion of 8-iso-PGF2alfa, collagen and elastic fibers content in alveolar septum and airways walls were performed. In airways walls, we also analyzed the number of MUC-5 positive cells and the number of macrophages. Results: The proteinase inhibitor EcTI was able to reduce the pulmonary mechanical alterations (Ers, Htis and Raw), alveolar septum disruption (Lm) and the BAL cell count (total cells, macrophages, neutrophils, lymphocytes and eosinophils) induced by elastase. Regarding the inflammatory response, EcTI also reduced the number of neutrophils and TNFalfa positive cells in both alveolar septum and airway walls, and also reduced the number of macrophages in alveolar septum. Considering the extracellular matrix remodeling, the proteinase inhibitor attenuated the volume fraction of collagen fibers, MMP-9 and MMP-12 positive cells in both alveolar septum and airway walls. Besides, in airway there were attenuation in the volume fraction of elastic fibers, and in the alveolar septa a decrease of the amount of the cells expressing TIMP-1. Regarding the oxidative stress response, EcTI reduced the volume fraction of isoprostane and the number of iNOS and eNOS positive cells in both airways walls and alveolar septa, Finally, EcTI reduced the number of MUC5ac positive cells in airway walls. Conclusions: The treatment with EcTI modulated lung mechanics and reduced inflammatory, remodeling and oxidative stress alterations induced by elastase. Although more studies need to be performed to elucidate the mechanisms involved in this process, we may considerate EcTI as a potential therapeutic tool for COPD management