No description available.

Inhibitor of Differentiation (ID) proteins are a family of four (ID1-4) bHLH transcription factors that lack the DNA binding domain. They act by forming dimers with other transcriptional regulators and inhibiting their interaction with DNA. They play a crucial role during embryonic development and later in the adulthood, their expression is mostly restricted to a few populations of stem cells. In the last decades, many authors have described their re-activation and participation in tumor development, angiogenesis and EMT although the results are still controversial. In the first chapter of this research work, the role of ID genes as prognostic markers in breast cancer was evaluated. We studied the mRNA expression of the four ID genes and four markers of EMT by qRT-PCR in a clinical series of 307 primary breast carcinomas previously stratified in immunophenotypes. In addition, the expression of all these genes was measured in breast cancer cell lines and mammospheres. Overexpression of at least one ID gene was found in 48.9% of the studied samples. ID1 and ID4 were overexpressed mostly in TNBL and HER2-enriched subtypes, whereas ID2 and ID3 were overexpressed more frequently in luminal tumors. High ID1 and ID4 was associated with larger tumor size, histological grade 3, presence of necrosis and vascular invasion, and poorer outcome. Multivariate analysis revealed that ID4 and vascular invasion were independent factors for DFS. Regarding EMT markers, high levels of SNAI1 were associated with the overexpression of the four ID genes. Additionally, ID1 overexpression was positively related to TWIST1, and the overexpression of ID2 and ID3 was more frequently paired with tumors that conserve CHD1 expression. In vitro studies showed high expression of the four ID genes in all cell lines. However, when mammospheres were formed, mRNA levels of ID genes decreased, in contrast to SNAI1 and TWIST1, which mostly increased. In the second chapter of this thesis, we aimed to (a) describe the mechanisms of action of a small molecule pan-ID antagonist, (b) define its main targets and (c) investigate potential pathways of acquire resistance. Treatment with AGX51 led to Id protein loss, increase in ROS accumulation, cell cycle arrest, and cell death in all tumor cell lines tested. Here, we used an antioxidant compound in different cell lines to demonstrate that ROS are the main responsible of cell death following treatment with AGX51. A model of cultured quiescent cells not expressing ID proteins served to show that the main target of AGX51 are these proteins. Experiments with AGX-derivatives also supported these results. Finally, three mutagenizing agents were used in order to generate mutations that confer resistance to treatment with AGX51. Treatment with ENU gave rise to two clones apparently resistant to AGX51 effects. Based on our in vitro and clinicopathological studies, we conclude that ID1 and ID4 may act as biomarkers of worse prognosis in patients with breast cancer, and seem to be involved in the initiation of EMT mechanism. Therefore, they are potential targets for the development of novel drugs. In line with this, AGX51 arises as a potent anti-ID compound that has anticancer effects.

No description available.

Protein phosphorylation is a reversible post-translational modification that is critical for the regulation of key cellular processes. It is estimated that two-thirds of all cellular proteins are phosphorylated, with more than 98% of those phosphorylation events occurring on Ser/Thr residues. Protein phosphorylation is mediated by protein kinases and reversed via dephosphorylation by protein phosphatases. Two protein phosphatases, phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), are thought to account for more than 90% of the total phosphatase activity in eukaryotic cells. PP2A is a highly conserved assortment of heterotrimeric holoenzymes responsible for the dephosphorylation of many regulated phosphoproteins. Substrate recognition and the integration of regulatory cues are mediated by B regulatory subunits that are complexed to the catalytic subunit (C) by a scaffold protein (A). Substrate recruitment of PP2A/B55α, the most abundant PP2A holoenzyme, was thought to be mediated by charge-charge interactions between the surface of B55α and its substrates. Challenging this view, we recently discovered a conserved SLiM (Short Linear Motif) [RK]-V-x-x-[VI]-R in a range of proteins, including substrates such as the retinoblastoma-related protein p107 and TAU (Fowle et al. eLife 2021;10:e63181). Here we report the identification of this SLiM in FAM122A, an inhibitor of B55α/PP2A, and analysis of the associated proteomic datasets that aided in identifying FAM122A, which can assist in the further identification of potential substrates and cellular pathways regulated by this phosphatase. The newly identified conserved SLiM is necessary for FAM122A binding to B55α in vitro and in cells. Computational structure prediction with AlphaFold2 predicts an interaction consistent with the mutational data and supports a mechanism whereby FAM122A uses the ‘SLiM’ in the form of a short α-helix to dock to the B55α top groove. In this model, FAM122A spatially constrains substrate access by occluding the catalytic subunit with a second α-helix immediately adjacent to helix-1. Consistently, FAM122A functions as a competitive inhibitor as it prevents the binding of substrates in in vitro competition assays and the dephosphorylation of CDK substrates by B55α/PP2A in cell lysates. Ablation of FAM122A in human cell lines reduces the rate of proliferation, the progression through cell cycle transitions, and abrogates G1/S and intra-S phase cell cycle checkpoints. FAM122A-KO in HEK293 cells results in the attenuation of CHK1 and CHK2 activation in response to replication stress. Overall, these data strongly suggest that FAM122A is a ‘SLiM’-dependent, substrate-competitive inhibitor of B55α/PP2A that suppresses multiple functions of B55α in the DNA damage response and in timely progression through the cell cycle interphase. In agreement with these findings, ectopic expression of B55α results in the downregulation of 14-3-3σ signaling mediated by ATM and ATR as determined by pathway analysis of phosphoproteomic datasets and a reduction of ATM signaling within the total proteome. Altogether, this work has significantly expanded our understanding of the PP2A/B55 SLiM, resulting from the characterization of FAM122A, a high-affinity substrate inhibitor, and enables future interrogation of novel substrates and signaling networks regulated by PP2A/B55α. / Biomedical Sciences

Despite the remarkable success of adoptive T cell therapy in the treatment of melanoma and hematological malignancies, therapeutic capacity in a broad range of solid tumours is impaired due to immunosuppressive events that render tumour-specific T cells unable to persist and kill transformed cells. To address some of the limitations of ACT in solid tumours, our laboratory has developed a therapeutic modality utilizing oncolytic virus, which expresses a tumour-associated antigen, known as an oncolytic viral vaccine (OVV), in combination with tumour specific central memory T cells. With this therapeutic approach (ACT), we can achieve robust in vivo expansion of transferred cells resulting in the complete and durable tumour regression in multiple solid murine tumour models. However, we demonstrate that the curative potential is lost when the tumour stage and burden increase as expanded transferred cells differentiate to a dysfunctional state resulting in the progressive decline in the tumour-specific CD8+ T cell response. Thus, we believe that restoring the T cell response in late-stage tumours will lead to enhanced curative potential of ACT in late-stage tumours. We have previously shown that HDACi, MS-275, can enhance the therapeutic capacity of a T cell-based therapy in an aggressive brain tumour model. In addition, concomitant delivery of MS-275 with ACT ensures durable cures through immunomodulatory mechanisms. Strikingly, concomitant delivery of MS-275, a class 1 histone deacetylase inhibitor (HDACi), with ACT in late-stage tumours completely restores the transferred T cell response to similar levels observed in early-stage tumours resulting in the complete regression of advance-stage tumours. Furthermore, MS-275 enhanced the proliferative capacity and tumour-specific cytotoxic function of transferred cells, independently of tumour stage, type and mouse strain. Interestingly, we did not observe a complete reversal of T cell dysfunction, but rather observed that MS-275 conferred unique properties to T cells as the expression of some markers typically associated with T cell dysfunction was enhanced in addition to persistence and proliferation capacity. Moreover, concomitant delivery of MS-275 also restored the therapeutic capacity of endogenously primed tumour-specific CD8+ T cells expanded by an OVV in late-stage tumours, demonstrating the potential for general use for MS-275 in T cell-based therapies. Our data suggests the use of HDACi may potentiate T cell-based immunotherapies to overcome tumour-mediated T cell dysfunction in advanced stage solid tumours. / Thesis / Master of Science in Medical Sciences (MSMS)

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24478号 / 医博第4920号 / 新制||医||1062(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 山本 洋介, 教授 近藤 尚己, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24496号 / 医博第4938号 / 新制||医||1064(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 滝田 順子, 教授 上野 英樹, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Antimikrobielle Resistenzen stellen eine weltweite Herausforderung dar und sind mit einer hohen Morbidität und Mortalität verbunden. Die Letalitätsrate durch multiresistente Keime steigt stetig an, weshalb die WHO im Jahr 2017 eine Prioritätenliste resistenter Keime erstellte, die die Entwicklung neuer Antibiotika vorantreiben soll. Diese umfasst vornehmlich gramnegative Bakterien, da diese aufgrund ihres Zellaufbaus sowie diverser Resistenzmechanismen besonders widerstandsfähig gegenüber dem Angriff vieler Antibiotika sind. Einige grampositive Keime (z.B. S. aureus) stehen ebenfalls auf dieser Liste und stellen eine große Herausforderung für die Medizin dar. Infolgedessen ist die Entwicklung neuer Antiinfektiva mit neuen Angriffspunkten gegen resistente Pathogene zwingend nötig, um mit bisherigen Resistenzen umgehen zu können. Die vorliegende Arbeit beschäftigt sich mit der Entwicklung und Synthese von kovalent (reversibel) bindenden Inhibitoren der Enoyl-ACP-Reduktase FabI (Staphylococcus aureus, Escherichia coli) und der Thiolase FadA5 (Mycobacterium tuberculosis). Beide Enzyme sind essenziell für das Überleben des jeweiligen Bakteriums. FabI ist ein wichtiges und geschwindigkeitsbestimmendes Schlüsselenzym der Fettsäuresynthese Typ II diverser Bakterien. Hierbei werden wichtige Phospholipide hergestellt, die für den Aufbau der Zellmembran nötig sind. Schiebel et al. ist es gelungen, einen potenten Inhibitor für den Erreger S. aureus sowie E. coli zu entwickeln und zu charakterisieren. Ausgehend von dieser Verbindung wurde eine Substanzbibliothek mit verschiedenen „warheads“ hergestellt. Hierbei wurde die Verknüpfung zwischen dem Pyridon-Grundgerüst und der elektrophilen Gruppe sowie die über den Ether verknüpften aromatischen Ringsysteme variiert. Diese Verbindungen wurden hinsichtlich ihrer inhibitorischen Aktivität am jeweiligen Enzym getestet. Anschließend wurde von Verbindung 32 und 33, die jeweils eine gute Inhibition des Enzyms aufweisen, der IC50-Wert gemessen. Beide Verbindungen weisen eine 50-prozentige Reduktion der Enzymaktivität im mittleren nanomolaren Bereich auf. Zusätzlich wurde Verbindung 32 in einem sogenannten „jump-dilution“-Assay auf kovalente Inhibition getestet. Durch dieses Experiment konnte eine kovalente Inhibition des Enzyms ausgeschlossen werden. Die Reaktivität der eingesetzten „warheads“ wurde gegenüber einem Tripeptid mittels eines LC/MS-Iontrap-Systems bestimmt. Die untersuchten Verbindungen zeigten keine signifikante Reaktion mit der im Tripeptid eingebauten nukleophilen Aminosäure Tyrosin, deren Nukleophilie bei dem pH-Wert des Tests (pH = 8.2 und 10.8) nicht hoch genug ist. Um einen Einblick in den Bindemodus der Verbindungen zu erhalten, wurden ferner Kristallisationsversuche durchgeführt. Die erhaltenen Kristallstrukturen zeigen, dass die Verbindungen mit dem gewünschten Bindemodus am Zielenzym binden, aber eine kovalente Modifizierung des Tyrosins146 durch die eingesetzten „warheads“ aufgrund der großen Entfernung (6 Å zwischen elektrophiler Gruppe und Tyrosin146), unwahrscheinlich ist. Zusätzlich wurden die physikochemischen Eigenschaften (Stabilität, Wasserlöslichkeit und logP) der Verbindung 32 sowie Verbindung 33 charakterisiert. M. tuberculosis ist der Erreger der global verbreiteten Infektionskrankheit Tuberkulose (TB), die zu den zehn häufigsten Todesursachen weltweit gehört. Das Bakterium kann das im menschlichen Körper vorkommende Cholesterol metabolisieren und nutzt dessen Abbauprodukte als wichtige Kohlenstoffquelle. Die Thiolase FadA5 ist bei diesem Abbau ein wichtiges Enzym und konnte als potenzielles innovatives Target für neue Antibiotika definiert werden. Durch Dockingstudien konnten zwei potenzielle Leitstrukturen als Inhibitoren der Thiolase FadA5 identifiziert werden. Im Rahmen dieser Arbeit wurden die vorgeschlagenen Strukturen mit dem gewünschten „warhead“ synthetisiert und hinsichtlich ihrer inhibitorischen Aktivität gegenüber dem Enzym untersucht. Die Zielverbindungen zeigen keine signifikante Hemmung sowie kovalente Bindung über die eingesetzten „warheads“ an die Thiolase FadA5. / Antimicrobial resistance poses a global challenge and is associated with high morbidity and mortality. The case fatality rate of infections caused by multidrug-resistant pathogens continues to be on the rise, causing the WHO to compile a priority pathogens list that is supposed to advance the development of new antimicrobial compounds. The list is mainly comprised of gramnegative bacteria, since these are especially resilient to many antibiotics. This is due to their cellular structure and various mechanisms of resistance. Some grampositive bacteria are also a danger to public health and are therefore part of this list. Consequently, there is an urgent need for the development of new antiinfectives with novel modes of action, so that the current resistance situation can be adequately addressed. This work is concerned with the development and synthesis of covalent reversible inhibitors of the enoyl-ACP reductase FabI (Staphylococcus aureus, Escherichia Coli) and the thiolase FadA5 (Mycobacterium tuberculosis). Both enzymes are critically important for the survival of the respective bacteria. FabI is an essential and rate determining enzyme of the type II fatty acid synthesis of various bacteria. A number of important phospholipids required for the cell membrane are biosynthesized via this metabolic pathway. Schiebel et al. were able to develop and characterize a potent inhibitor for S. aureus and E. Coli. Using this compound as a starting point, a library of compounds carrying various “warheads” was synthesized. Further structural variations were introduced by using different linkers between the pyridone scaffold and the electrophilic group as well as diverse aromatic rings connected via the ether bridge. These compounds were assayed concerning their inhibitory activity at the respective enzyme. Of these, substances 32 and 33 showed good inhibition of the enzyme, prompting the determination of the IC50 values. The two substances were able to reduce enzymatic activity by 50% at nanomolar concentration levels. In addition, substance 32 was characterized concerning its ability to covalently inhibit its molecular target by means of the so-called jump dilution assay. This experiment showed no covalent inhibition of the target enzyme. The individual reactivity of the warhead moieties present in the library was determined against a synthetic tripeptide by using a LC/MS iontrap system. All the examined compounds showed no reaction with the nucleophilic amino acid tyrosine contained in the tripeptide at significant levels, which indicates that its nucleophilicity is insufficient at the pH of the assay (pH = 8,2 and 10,8, respectively). Crystallization experiments were conducted to ascertain the binding mode of the compounds. The crystal structures showed the substances binding to the enzyme in the desired pose, yet a covalent modification of tyrosine146 remains unlikely due to the large distance (6 Å) between the electrophilic moiety and the amino acid. Additionally, some physicochemical properties (Stability, aqueous solubility and logP) of compounds 32 and 33 were characterized. M. tuberculosis is the causative pathogen of the globally occurring infectious disease tuberculosis, which belongs to the 10 most frequently occurring causes of death worldwide. The germ is able to metabolize the cholesterol present in the human body and uses its degradation products as an important carbon source. The thiolase FadA5 is involved in this metabolic pathway and was identified as a potentially innovative target for novel antibiotics. Docking studies enabled the identification of two potential lead structures for inhibitors of FadA5. In this work, the proposed structures carrying the desired warheads were synthesized and characterized concerning their inhibitory activity at the target enzyme. The target compounds showed no significant inhibition or covalent binding to FadA5.

Die Rheumatoide Arthritis ist eine häufig auftretende, chronisch entzündliche Systemerkrankung und wird bei bis zu einem Drittel der Patienten mit einer T-LGL-Leukämie diagnostiziert. Wie häufig klonale T-LGL-Zellen bei Patienten mit Rheumatoider Arthritis auftreten, ist ungeklärt. Ziel dieser Studie war es, die Oberflächenantigene der T-Lymphozyten in einem Patientenkollektiv mit Rheumatoider Arthritis zu bestimmen. Der Fokus lag dabei auf der Prävalenz von klonalen T-LGL-Zellexpansionen und möglichen Risikofaktoren. Hierfür wurden zwischen November 2013 und August 2015 527 Patienten mit Rheumatoider Arthritis mittels Durchflusszytometrie untersucht. Zur Bestätigung der Klonalität erfolgte bei Patienten mit auffälligem Immunphänotyp eine PCR (Polymerase-Kettenreaktion)-Analyse. Bei 19 Patienten konnte eine klonale T-LGL-Zellexpansion festgestellt werden, was einer Prävalenz von 3,6% entspricht. Das Auftreten von klonalen T-LGL-Zellen war mit einer TNFα-Inhibitoren-Therapie (p=0,01) und deren Dauer assoziiert (p=0,01). Ob die klonalen T-LGL-Zellen Ausdruck der Autoimmunerkrankung oder Vorläuferzellen einer T-LGL-Leukämie sind, bleibt offen. Die Patienten werden mit einer klonalen T-LGL-Zellexpansion unklarer Signifikanz beschrieben. / Rheumatoid arthritis is a common, chronic inflammatory systemic disease and is diagnosed in up to a third of patients with T-LGL leukemia. The frequency of clonal T-LGL-cells in patients with rheumatoid arthritis is unknown. The aim of this study was to determine surface antigens of T-lymphocytes in a collective of patients with rheumatoid arthritis. The focus was on the prevalence of clonal T-LGL cell expansion and possible risk factors. Therefore, 527 patients with rheumatoid arthritis were examined using flow cytometry between November 2013 and August 2015. To confirm clonality, a PCR (polymerase chain reaction) analysis was carried out in patients with an aberrant immune phenotype. A clonal T-LGL-cell expansion was found in 19 of those patients, which corresponds to a prevalence of 3,6%. The occurrence of clonal T-LGL-cells was associated with TNFα inhibitor therapy (p=0,01) and its duration (p=0,01). Whether the clonal T-LGL-cells are an expression of the autoimmune disease or precursor cells of T-LGL leukemia remains unclear. Therefore, those patients are described with a clonal T-LGL-cell expansion of uncertain significance.

Simultaneous maternal indirect blood pressure measurements, electronic fetal heart rate monitoring, and ultrasonographic biophysical profile testing were used to assess maternal-fetal homeostasis in gravid ewes during gestational days 127 to 134 (term 146), during a 72-hour fast, and during treatment with thromboxane synthetase inhibitors CGS13080 and CGS12970. Seventy-five percent of the ewes (12 of 16) developed clinical signs of ovine pregnancy-induced hypertension, including maternal hypertension and fetal depression. In three untreated hypertensive ewes, pregnancy was terminated by spontaneous premature delivery, and one maternal death occurred after an eclamptic seizure. All nine ewes treated with one of the two thromboxane synthetase inhibitors responded to therapy with decreases in blood pressure and resolution of fetal depression. These nine ewes completed gestation, and were delivered at term. These data indicate that therapy with thromboxane synthetase inhibitors in this animal model of preeclampsia results in profoundly beneficial effects and suggest that further studies of thromboxane synthetase inhibitors are warranted in preeclampsia.