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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22733号 / 医博第4651号 / 新制||医||1046(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 萩原 正敏, 教授 岩田 想, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway. / PHD / Malaria is a severe and potentially fatal mosquito-borne disease. The continuous emergence of insecticide-resistant mosquitoes and drug-resistant parasite strains necessitates the development of novel antimalarial agents, notably those that engage new targets in these organisms. Herein we present three projects in which the synthesis and characterization of new malaria insecticide and therapeutic candidates are described. Our aim in the first project was to synthesize acetylcholinesterase (AChE) inhibitors as potential mosquitocides to be deployed on insecticide-treated nets. Three different classes of compounds were synthesized and characterized. Their potency to inhibit the wild-type and insecticide-resistant mosquito AChE, and their corresponding mosquito toxicities were assessed. Mosquito-toxic compounds were identified, but they did not show appreciable selectivity between mosquito and human AChEs. The second project was directed toward finding the biological target of a known antimalarial drug; mefloquine (MQ). Numerous different MQ analogs were synthesized, and their potency was assessed in two biochemical assays. The results of this study strongly suggest that MQ kills malaria parasites by preventing them from ingesting the red blood cell hemoglobin. The third project was concerned with the optimization of a compound (MMV008138) that kills malaria parasites by preventing it from synthesizing a key biochemical building block (IPP). Several new compounds were prepared that had similar antimalarial activity to MMV008138, of which many have better potential to serve as antimalarial drugs. In addition, these studies provided valuable insights for the design of further improved analogs.

La molécule UM171 amplifie les greffons de cellules souches hématopoïétiques. Cela permet l’utilisation de greffons de cellules souches provenant du sang de cordon ombilical pour des greffes chez les patients adultes, notamment dans les cas de cancers du sang. Bien que le mode d’action biologique d’UM171 soit connu, la protéine à laquelle se lie UM171 reste indéterminée. Le but de ce projet est d’identifier cette protéine-cible. Afin d’atteindre cet objectif, cette étude mise sur la conception, la synthèse, puis l’utilisation de sondes d’affinité. Dans ce mémoire, deux types de sonde sont présentées : les sondes d’affinité non covalentes et les sondes d’affinité photoréactives. Dans un premier temps, une sonde non covalente est synthétisée. Cette sonde, très active, est ensuite utilisée dans un essai de chimio-protéomique. Elle permet d’identifier les protéines KBTBD4, RCOR1 et HDAC2 par buvardage de western. Dans un deuxième temps, une sonde photoréactive est conçue, puis synthétisée. Considérant l’hypothèse de la présence d’un acide carboxylique dans le site actif de la protéine-cible, cette sonde exploite une fonctionnalité 2,5-diaryltétrazole comme groupement photoréactif sélectif aux acides carboxyliques. Afin de maximiser l’activité de cette sonde, celle-ci est développée grâce à l’étude de plusieurs analogues, dont la synthèse est également rapportée. Dans un troisième temps, la sonde photoréactive est utilisée dans deux essais de chimio- protéomique. Ces essais ont permis d’identifier la protéine CUL3 par spectrométrie de masse. La protéine CAND1 a également été identifiée, de façon moins significative. / The small molecule UM171 amplifies hematopoietic stem cells grafts. This allows the use of umbilical cord blood stem cells grafts for transplants in adult patients, for example in the case of blood cancer. Although the biological mode of action of UM171 is known, the protein to which UM171 binds remains undetermined. The aim of this project is to identify this target protein. To achieve this goal, this study focuses on the design, synthesis, and use of affinity probes. In this thesis, two types of probes are presented: non-covalent affinity probes and photoreactive affinity probes. First, a non-covalent probe is synthesized. This probe revealed to be very potent and is used in a chemoproteomics assay. The proteins KBTBD4, RCOR1 and HDAC2 are identified by western blotting. Second, a photoreactive probe is designed, and then synthesized. Assuming the presence of a carboxylic acid in the active site of the target protein, this probe exploits a 2,5-diaryltetrazole functionality as its photoreactive group selective to carboxylic acids. To maximize the activity of this probe, it is designed by studying several analogues, the synthesis of which is also reported. Third, the photoreactive probe is used in two chemoproteomics assays. These led to the identification of the protein CUL3 by mass spectrometry. The protein CAND1 was also identified, less significantly.