京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23425号 / 医科博第130号 / 新制||医科||9(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 萩原 正敏, 教授 戸井 雅和, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Cryogenic transmission electron microscopy (cryoEM) has become an increasingly common tool for determining structures of proteins and protein complex at near atomic resolution. We seek to determine the structure of p97 by cryoEM using an affinity capture approach that employs a family of novel synthetic lipids bearing water soluble PEG units and known high affinity inhibitor molecules at the distal end of the polymer. A library of inhibitor modified affinity lipopolymers of 5000 KD PEG molecular weights were synthesized. The inhibitor modified lipid coated grids were used to capture p97. The reconstruction of p97 revealed the structure at dimeric state at 3.64 Å and monomeric state at 4.33 Å. A PEG unit composed of 20000 KD molecular weight based polyrotaxane containing NTA ligand as affinity tag has been synthesized, used to concentrate 6x-his tagged p97 on TEM which also enabled to see all 3D orientation of the target particles and an initial model of 10.64 Å resolution of p97 structure was resolved. </p>

Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

Platinum-based anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin, have been approved for clinical use worldwide for decades. Despite their enormous success, their widespread application is hindered by either cross-resistance or toxic side effects, including nephrotoxicity and neurotoxicity. The need to overcome these drawbacks has stimulated the search for new platinum-based drugs. This dissertation will start with the accidental discovery of cisplatin, followed by an introduction of other platinum-based anticancer agents, including the action mechanism, general structures, and development history. Picoplatin is a newer generation of platinum-based anticancer agent. The bulky 2-methylpyridine as a non-leaving group on picoplatin could reduce the detoxification effect caused by thiol-containing species, such as glutathione and metallothionein, thus may grant picoplatin the ability to overcome cisplatin resistance. A convenient synthesis route for picoplatin derivatives has been developed. 11 new picoplatin derivatives have been designed by varying the bulkiness of the non-leaving amine group. All complexes have been characterized by different instrumentations, including MS, 1H NMR, 13C NMR, 195Pt NMR, HMQC, X-ray crystallography, and elemental analysis. Different bioassays, such as DNA binding, cell viability, and cellular accumulation, have been applied to evaluate their efficacy on cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780cis. The newly designed picoplatin derivatives show comparable efficacy with that of picoplatin and less resistance compared with cisplatin. The study of picoplatin derivatives laid the foundation toward the research of bifunctional platinum-based anticancer agents by incorporating histone deacetylase (HDAC) inhibition. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are a pair of important enzymes in epigenetic regulation. They work in harmony to acetylate and deacetylate histone lysine residues, resulting in a more relaxed or more condensed chromatin structure, respectively. HDAC has been found to be overexpressed in some cancer cells. Since 2006, 5 HDAC inhibitors (HDACi) have entered clinical use for cancer treatment. 19 new HDACi with additional coordination sites on the phenyl cap have been designed, synthesized, and evaluated. A few of the new HDACi show comparable or even better HDAC inhibition than that of Vorinostat (SAHA, the first FDA approved HDACi). A logical design would involve the installation of HDACi on the platinum center as a non-leaving group ligand. When the bifunctional drug reaches the cancer cell, the synergistic effect could be maintained as the relaxed chromatin structure makes DNA more susceptible to be attacked by the platinum centers, thus increase the anticancer activity and possibly selectivity toward cancer cells. 6 Pt-HADCi conjugates have been designed and synthesized. Dual functions of the new Pt-HDACi have been confirmed by DNA electrophoresis assay and HDAC inhibition assay. One of the Pt-HDACi (CF-101) shows comparable cytotoxicity with cisplatin and less resistance, which could be used as the lead compound for further structural modification and in vivo studies.

<p>Since 1981, HIV/AIDS has affected over 70 million individuals worldwide. Due to the incorporation of Combination Antiretroviral Therapy (cART), this deadly virus has now become a manageable chronic illness with a reduction in mortality and morbidity rates. Combination therapy targets multiple stages of the HIV replication cycle including fusion, entry, reverse transcription, integration, and maturation. The HIV-1 protease enzyme is responsible for cleavage and processing of viral polyproteins into mature enzymes and is a common therapeutic target for inhibition of HIV. To date, there have been many protease inhibitors approved by the FDA and introduced into the market. However, mutations within the protease enzyme has rendered some of these inhibitors ineffective. This has led to an ever-growing need to develop novel protease inhibitors to combat drug resistance through mutations. Described herein is the design, synthesis, and biological evaluation of HIV-1 protease inhibitors featuring a novel hexahydropyrrolofuran (HPF) bicyclic scaffold as a P₂ ligand to target binding interactions with Asp29 and Asp30. The HPF ligand provides a molecular handle that allows for further structure-activity discoveries within the enzyme. The HIV-1 protease inhibitors discussed feature carbamate, carboxamide, and sulfonamide derivatives which displayed good to excellent activity.</p>

Les microARN (miARN) constituent une classe de petits ARN non-codants qui jouent un rôle clé dans la régulation de l’expression des gènes au niveau post-transcriptionnel. De nombreuses études ont démontré que la surexpression de certains miARN est liée au développement de plusieurs types de cancer. C’est pour cette raison les miARN représentent une nouvelle classe de cibles thérapeutiques à haute potentielle. Dans ce contexte, l’objectif de mon travail de thèse était la découverte de nouveaux inhibiteurs de la production de miARN oncogène. Dans ce but, j’ai suivi deux approches, différentes mais complémentaires : (i) le criblage d’une petite librairie de composés et (ii) la conception et la synthèse de nouveaux conjugués en tant que ligands de précurseurs de miARN (pré-miARN). En particulier, nous avons ciblé les miARN-372 et -373 qui sont oncogènes dans plusieurs cancers, tels que le cancer gastrique. Nous avons ainsi démontré que certains des composés criblés ou synthétisés sont capables de se lier efficacement à la structure secondaire en tige-boucle de pré-miARN avec une haute affinité, conduisant à l’inhibition de la production des miARNs correspondants. Par ailleurs, nous avons montré que certains composés possèdent une activité anti-proliférative spécifique pour les cellules de cancer gastrique et que cette activité est directement liée à une diminution de la production de miARN ciblés et au rétablissement de la traduction de ARN messenger / MicroRNAs (miRs) are a class of small non-coding RNAs that act as regulators of gene expression at the post-transcriptional level. Increasing evidence has indicated that the deregulation of miR expression is linked to various human cancers and therefore, miRs represent a new class of potential drug targets. In this context, my PhD project focused on the discovery of new inhibitors of oncogenic miRs production. Toward this aim, two different but complementary approaches were followed: (i) the screening of small libraries of compounds and (ii) the design and synthesis of new classes of conjugates as binders of miRNA precursors (pre-miRs). In particular, we focused our attention on miR-372 and miR-373, two oncogenic miRs overexpressed in various cancers, such as gastric cancer. We showed that some of the screened or of the newly synthesized compounds are able to efficiently bind to stem-loop structured precursors of the targeted miRs with high affinity, thus inhibiting the production of their corresponding mature miRs at the level of Dicer cleavage. Moreover, we found compounds bearing a specific anti-proliferative activity in gastric cancer cells overexpressing targeted miRs and this activity is directly linked to a decrease in the production of oncogenic miR-372 and -373 and to the restoration of normal mRNA translation.

Auxin is one of the most important plant hormones, which provides development of a plant. PIN1 and PIN7 proteins belong to the PIN family of transporters which is among the most important auxin efflux carriers. This thesis deals with the of AtPIN1 and AtPIN7 auxin efflux carriers (from Arabidopsis thaliana) in human embryonic kidney 293 cell line. Biological activity of these proteins was tested by using radiolabeled auxins accumulation. Further inhibitors of auxin transport have been tested - NPA, CHPAA and BFA.

Influenza viruses cause respiratory illnesses which can vary in severity depending on the strain of the virus, as well as the age and health condition of the host. Influenza remains a major threat to public health due to its nature prone to suffer mutations. As a result, vaccines have to be reformulated annually and new strains may cause sporadic global pandemics. Furthermore, the recent emergence of resistant strains of the virus against the current standard of care (oseltamivir and zanamivir) underlines the need of novel anti-influenza therapeutics. The aim of this dissertation work is to contribute to the discovery of new anti-influenza inhibitors either by rational drug-design and optimization of oseltamivir structure, or by developing screening assays suitable for the discovery of novel inhibitors of the enzymes neuraminidase or RNA-polymerase. Scheme 1. Overview of the strategy used for the development of new anti-influenza therapeutics. The dashed arrows indicate the inhibitors that were converted into probes and their corresponding target enzymes Two main modification points were explored for the improvement of oseltamivir properties (Scheme 1); modifications at carbon C-3 aimed to overcome oseltamivir resistance caused by common mutations like H274Y, meanwhile modifications at carbon C-5...

Vitamin B1 is an indispensable co-factor for various enzymes inter alia in the Krebs cycle, pentose phosphate pathway, nucleotide and amino acid synthesis. Due to its importance in metabolism, proteins involved in the synthesis of vitamin B1 have been identified as potential drug targets. Thiamine monophosphate kinase (ThiL), catalyses the last reaction in the pathway, the ATP dependent phosphorylation of thiamine monophosphate (TMP) producing thiamine pyrophosphate (TPP) the active and co-factor form of vitamin B1. In this study, thiamine monophosphate kinase from Mycobacterium tuberculosis (MtbThiL, ~36 kDa) was produced as an N-terminally His6-tagged fusion protein, purified by affinity and size exclusion chromatography, and crystallised. Hexagonal MtbThiL crystals belonged to space group P6122. Molecular replacement revealed a symmetric homodimer with a single monomer occupying the asymmetric unit. Analysis of the structure showed that each subunit of MtbThiL has an ATP and TMP binding site and is structurally related to other ThiL enzymes. Ten lead compounds were identified from compound databases as potential ThiL inhibitors, and oxythiamine was chosen for further study. The binding affinities of oxythiamine and TMP to MtbThiL were determined by isothermal titration calorimetry and a pyruvate kinase-lactate dehydrogenase enzyme assay, which revealed that the binding affinity for oxythiamine by MtbThiL is lower than the substrate TMP. / Dissertation (MSc)--University of Pretoria, 2020. / Biochemistry / MSc / Unrestricted

Auf der Basis des Pilling-Bedworth-Verhältnisses für die Charakterisierung der Oxidationsneigung wurden die für die Herstellung von Magnesiumgussteilen in Sandformverfahren relevanten Einflussfaktoren, Formstoff- und Werkstoffkennwerte erfasst. Ausgehend davon werden geeignete Schutzprinzipien und technologische Maßnahmen formuliert. Die vorgeschlagenen Schutzkomplexe (Borsäure und Harnstoff) vermeiden die Oxidation des Magnesiums, reduzieren deutlich den Wasserbedarf und verbessern die Fließeigenschaften des Formstoffes. Die Zusätze sind fluor- und schwefelfrei, was geringe Emissionen und bessere Arbeitsbedingengen ermöglicht. Im Rahmen der Arbeit erfolgte eine umfassende Entwicklung praxistauglicher bentonitgebundener und kalthärtender organischer Formstoffsysteme und Schlichten. Die Einsetzbarkeit der Formstoffe für die moderne Formtechnik wurde nachgewiesen. Die erreichbaren Werkstoffeigenschaften entsprechen den internationalen Normen, hervorzuheben ist die hohe Oberflächenqualität der Gussteile. Die erzielten Ergebnisse wurden auf die realen Bauteile und Herstellungsprozesse, wie z.B. zum Ablösen des Natursandformverfahrens und für die Herstellung eines 4-Zylinder Motorblockes, erfolgreich übertragen. Durch die Realisierung der dargestellten Innovationen im Bereich der Sandformverfahren werden neue Impulse zur Verbesserung der Magnesium-Produktion und damit der internationalen Wettbewerbsfähigkeit im Fahrzeugbau gegeben.