Le sujet de cette thèse porte sur la synthèse d'inhibiteurs spécifique de la Sérine-Thréonine phosphatase I (PP1). Un criblage de la chimiothèque de l'institut Curie, réalisé par l'équipe du Dr. Popov a permis d'identifier une 3-aryl-1(2H)isoquinolone, qui perturbe la dynamique des microtubules et qui s’est ensuite avéré être un inhibiteur sélectif de PP1. Dans une première partie, nous avons mis au point une nouvelle méthodologie de synthèse de ces composés hétérocycliques par une réaction tandem d’aminocarbonylation-cyclisation pallado catalysée. L’étude d’une seconde voie de synthèse de ces composés a été étudié par réaction d'arylation direct d'une 1(2H)isoquinolone. Dans le but de trouver d’autres hit, ligand de cette phosphatase, nous avons tenté de développer un test de triple hybride chimique, en collaboration avec la société Hybrigenics. Ce test est basé sur l’interaction de notre inhibiteur hit avec la phosphatase PP1. Pour cela, nous avons synthétisé une sonde à partir de la molécule hit initiale. La deuxième partie a trait à un développement de chimie médicinal pour optimiser le hit initial. Des dérivés de très bonne sélectivité pour l’enzyme cible ont été préparés. / This PhD thesis deals with the synthesis of serine threonine phosphatase I (PPI) inhibitors. This project started with the screening of the Institut Curie’s Library carried out by Dr. Popov team. They identified a 3-aryl-1(2H)isoquinolone (hit molecule) which strongly disturbs the microtubules dynamics. In the first part, we designed an original methodology to prepare those heterocycles, though a tandem palladium catalyzed aminocarbonylation/cyclization reaction. Then, we studied the direct arylation reaction to obtain the desired scaffold. In collaboration with Hybrigenics, we synthesize a probe for a triple hybrid system, based on the specific interaction of the hit molecule with its target PPI. Thanks to this system, one could identify new inhibitors of the targeted phosphatase protein. Eventually, a library of isoquinolones derivatives was synthesized. During the invitro tests, some of those molecules proved to be very specific for the serine threonine phosphatase I.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

La phosphorylation de protéines correspond à l’addition covalente d’un groupement phosphate (PO4 3-) par une protéine kinase sur un substrat. Cette réaction est réversible : la déphosphorylation est catalysée par des protéines phosphatases. Chez S. aureus, la sérine/thréonine kinase Stk1 phosphoryle des acides aminés sérines et thréonines et a été montrée impliquée dans la régulation de la virulence du pathogène : nous avons approfondi les connaissances sur ce mécanisme en identifiant trois nouveaux substrats et en étudiant les effets de la phosphorylation sur leur activité : - l’enzyme LuxS, responsable de la synthèse de l’AI-2 impliqué dans la communication intra bactérienne, voit son activité enzymatique drastiquement diminuée en étant phosphorylée par Stk1 sur un site thréonine unique (T14) ; - le régulateur CcpA, dont la fixation sur l’ADN module l’expression de nombreux gènes de virulence, est phosphorylée par Stk1 sur deux sites (T18 et T33) et cette phosphorylation diminue l’affinité de la protéine régulatrice CcpA pour l’ADN de ses gènes cibles ; - l’élément réponse du système à deux composants SaeR est phosphorylé sur deux sites thréonines (T87 et T192) de la région impliquée dans l’affinité de SaeR pour l’ADN. Les rôles de la kinase Stk1 sont donc multiples et liés à la régulation de la virulence de S. aureus. Les autres voies mettant en jeu la phosphorylation de protéines bactériennes, comme les systèmes à deux composants ou le système CcpA/HPr, sont couplées à cette phosphorylation par la sérine/thréonine kinase : ces résultats soulignent à la fois la diversité et la complexité de la régulation des mécanismes responsables de la virulence de S. aureus / Protein phosphorylation consists in the catalyzed addition of a phosphate group on a substrate. This reversible reaction is ensured by both kinase and phosphatase proteins. S. aureus is a human prokaryote pathogen and a part of its virulence is known to be regulated by the serine/threonine kinase Stk1, which phosphorylates serine or threonine residues of its substrates. We investigated the mechanisms of this virulence regulation and newly identified three substrates of Stk1: the quorumsensing LuxS protein, the catabolite carbon protein CcpA and the two components system response element SaeR. LuxS is phosphorylated on a unique threonine residue in position 14 and phosphorylation dramatically influences its enzymatic activity on AI-2 production. CcpA phosphorylation on two threonine residues in the DNA-binding region of the protein (T18 and T33) decreases the affinity of the protein for its targeted DNA sequences. Besides, Stk1 also phosphorylates the response element SaeR on two threonine residues (T87 and T192) in the DNAbinding region. Therefore Stk1 kinase plays numerous roles in S. aureus virulence regulation and the complexity of this regulation pattern increases when considering that three of the phosphorylation pathways in prokaryotes are crossed over: the two components system phosphorylation, the HPr/HPrK system and the serine/threonine kinase proteins phosphorylation. These results highlight the need to focus on Stk1 as a key element in the complexity of virulence regulation in S. aureus

Orientadores: Sandra Martha Gomes Dias, Marília Meira Dias / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T09:41:29Z (GMT). No. of bitstreams: 1 Ascencao_CarollineFernandaRodrigues_M.pdf: 4713312 bytes, checksum: b65183d96535d66661af745a562f2d58 (MD5) Previous issue date: 2014 / Resumo: A proliferação celular comanda os processos de embriogênese e de crescimento do organismo, sendo essencial para a correta função de vários tecidos adultos. Apesar de ser importante para a homeostase do organismo, a sua desregulação compõe a força motriz do desenvolvimento tumoral. Somente nos últimos vinte anos começou a ser evidenciada a relação entre as vias de tradução de sinais estimuladas por fatores de crescimento e a reorganização da atividade metabólica, a qual precisa priorizar a biossíntese e o aumento da biomassa, processos essenciais para a divisão celular. Em células tumorais, o consumo de glutamina é aumentando concomitante ao aumento da atividade de glutaminase. Três isoenzimas de glutaminase são expressas na maioria dos tecidos (liver-type glutaminase, kidney-type glutaminase e glutaminase C), todavia pouco se sabe sobre a necessidade específica de cada uma delas para o metabolismo tumoral. Vários artigos recentes têm definido o papel da glutaminólise, ou metabolismo da glutamina e seus subprodutos, na ativação da mTOR. Neste sentido é uma hipótese válida imaginar que mTOR possa contra-regular glutaminase. Desta maneira, resolvemos investigar se mTOR atua na regulação da atividade de glutaminase. Para tanto, realizamos knockdown estável de PTEN em células MDA-MB 231 e verificamos que não o mesmo afetou os níveis protéicos de GAC e KGA, assim como não houve mudança na localização subcelular das isoformas. Cinética enzimática da fração mitocondrial desta linhagem revelou que o knockdown de PTEN levou à uma diminuição do KM da enzima sem alteração de Vmax. De acordo, o tratamento com rapamicina, inibidor da mTOR, elevou o KM para os níveis detectados nas células controles. A atividade de glutaminase de lisado total de MDA-MB 231, NIH 3T3, IMR90 e BJ5TA foi afetada pelo tratamento com rapamicina conforme julgado por ensaios de dose e tempo resposta. Mais, ensaios de privação de glicose, glutamina e de fatores de crescimento levaram à inibição de mTOR e concomitante redução da atividade de glutaminase. Somado a isso, o knockdown estável de TSC2 em MDA-MB 231 e BJ5TA, assim como o knockout de TSC2 em MEF, promoveu superestimulação de mTOR e foi capaz de aumentar a atividade de glutaminase. Dosagem de atividade de glutaminase de células MDA-MB 231 com knockdown de GAC, KGA ou GAC/KGA tratadas com rapamicina indicaram que mTOR possa agir em ambas as isoformas. Curioso foi que apenas células shGAC e shGAC/KGA apresentaram redução da fosforilação de S6K em Thr389 indicando que GAC ou o metabolismo de glutamina via esta isoforma, possa contra-regular mTOR. Em adição, na comparação entre PC3 e DU145, verificamos que DU145 apresentou maior expressão de GAC, maior consumo de glutamina, maior dependência de glutamina em seu crescimento, maior sensibilidade ao inibidor de glutaminase, BPTES, e por fim, se mostrou mais responsiva à metformina, ativador indireto de AMPK. A ativação de AMPK por metformina, um conhecido sensor de estresse energético, mostrou diminuir a atividade de glutaminase em célula de tumor de próstata, DU145, indicando uma potencial ação de AMPK na atividade de glutaminase / Abstract: Cell proliferation is crucial for embryogenesis and organism growth, being also essential for the proper function of several adult tissues. Although important for the homeostasis of the organism, its deregulation composes the driving force of tumor development. In the past twenty years the relationship between the processes of signal translation stimulated by growth factors and the reorganization of metabolic activity has become more evident. Growing cells need to prioritize the biosynthesis and biomass increase, processes essential for cell division. In tumor cells, the glutamine consumption is increased concurrently with the increasing in the glutaminase activity. Three glutaminase isoenzymes are expressed in most tissues (liver- type glutaminase, kidney -type glutaminase and glutaminase C), but not much is known about the necessity of each isoform for the tumor metabolism. Several recent papers have defined the role of glutaminolysis or glutamine metabolism in mTOR activation. So it is a valid hypothesis to speculate that mTOR can counter-regulate glutaminase. Thus, we decided to investigate whether mTOR can control glutaminase activity. To this end, we have made MDA - MB 231 cells stably knocked down for PTEN and verified no alteration in KGA and GAC protein levels, as well as there was no change on their subcellular location. Enzyme kinetics of the MDA-MB 231 mitochondrial fraction revealed that PTEN knockdown led to a decrease in the KM of the enzyme without changing Vmax. Accordingly, the treatment with rapamycin (mTOR inhibitor), led to an increase in KM back to the level detected in control cells. The glutaminase activity of MDA - MB 231, NIH 3T3, IMR90 and BJ5TA total cellular lysates was also affected by rapamycin treatment in a dose- and time-response fashion. Moreover, glucose, glutamine and growth factors deprivation promoted mTOR inhibition and concomitant reduction on glutaminase activity. Glutaminase activity of MDA-MB 231 cells knocked down for GAC, KGA or GAC/KGA and treated with rapamycin indicated that mTOR can regulate both isoforms. Curiously, it was only on GAC or GAC/KGA knocked down cells that we observed a decrease in S6K Thr 389 phosphorylation, which could indicate that GAC or the GAC dependent-glutamine metabolism is a specific mTOR counter-regulator. Accordling, stable TSC2 knockdown in MDA-MB 231 and BJ5TA, as well as TCS2 knockout in MEF cells, promoted overstimulation of mTOR and increasing on glutaminase activity. Moreover, a comparison between PC3 and DU145 revealed that DU145 has higher GAC expression, greater consumption of glutamine, is more dependent on glutamine for its growth, more sensitive to the inhibitor of glutaminase, BPTES, and more responsive to metformin, an indirect AMPK activator. The activation of AMPK by metformin, a known energy stress sensor, led to a decreased glutaminase activity in the prostate tumor cell line DU145 indicating a potential role of AMPK on glutaminase activity / Mestrado / Genetica Animal e Evolução / Mestra em Genética e Biologia Molecular

La capacidad de Mycobacterium tuberculosis para sobrevivir dentro del macrófago contribuye grandemente a su patogenicidad, latencia y persistencia durante la infección. Este bacilo induce alteraciones en el ambiente intrafagosomal e inhibe la maduración del fagosoma, favoreciendo su supervivencia intracelular. M. tuberculosis PknG secuestra al macrófago precisamente al evitar la fusión fagosoma-lisosoma. En este sentido, PknG representa una familia de dianas novedosas para enfrentar la necesidad de nuevos antimicrobianos para la tuberculosis latente. Aquí, apuntamos a: (i) elucidar la base estructural-molecular del ATP y Mg2+ como cofactores de PknG; (ii) caracterizar los parámetros cinéticos que gobiernan la formación del complejo PknG:ATP; e, (iii) identificar péptidos capaces de unirse a PknG para investigar experimentalmente su interactoma usando enfoques combinatorios como “Phage Display”. Nuestros resultados confirman que PknG se une exclusivamente al ATP con una constante de disociación (KD) de 108.8  22.9 µM. El Mg2+ estabiliza térmicamente a PknG de forma ATP-dependiente. Análisis de estado pre-estacionario muestran que la unión y disociación del ATP es rápida en el complejo PknG:ATP. Usando PknGN-Ext, TPR resolvimos la estructura cristalina en el estado unido al ADP mientras que demostramos que el ATP imposibilita la cristalización. Los análisis bioinformáticos de las librerías enriquecidas por Phage Display identificaron 57 potenciales peptidos que interactuarían con PknG. Una comparación cercana con el proteoma de M. tuberculosis proporcionó un subconjunto de 20 proteínas que podrían interactuar con PknG. Nuestros resultados confirmaron cinco proteínas asociadas a PknG previamente reportadas: PknG, DnaK chaperona, transportador ABC Rv1747, Proteína Ribosomal L23 y Factor de Elongación Tu, resaltando la validez de nuestra plataforma para descubrir el interactoma de PknG. Así, nuestros resultados revelan interacciones proteína-proteína putativas que podrían participar en la supervivencia micobacteriana, mientras que también proporcionan bases sólidas para desarrollar drogas antituberculosas al interrumpir estas interacciones o explotar estos peptidos tipo compuesto líder. / The ability of Mycobacterium tuberculosis to survive inside the macrophage greatly contributes to its pathogenicity, latency and persistence during infection. This bacillus induces alterations in the intraphagosomal environment and inhibits phagosome maturation, thus promoting mycobacterial survival. M. tuberculosis PknG hijacks the macrophage precisely by avoiding phagosome-lysosome fusion. In this sense, PknG represents a family of novel targets to cope with the need for new antimicrobials for latent tuberculosis. Here, we aimed to: (i) elucidate the structural-molecular basis of ATP and Mg2+ as PknG cofactors; (ii) characterize the kinetic parameters governing PknG:ATP complex formation; and, (iii) identify PknG-binding peptides to experimentally query PknG’s interactome using combinatorial approach such as Phage Display. Our results confirm that PknG exclusively binds to ATP with a dissociation constant (KD) of 108.8  22.9 µM. Mg2+ thermally stabilizes PknG in an ATP-dependent manner. Pre-steady-state analyses show that ATP binding and dissociation are rapid in the PknG:ATP complex. Using PknGN-Ext, TPR we solved the ADP-state crystal structure while showing that ATP precludes crystallization. Phage Display and bioinformatic analyses identified 57 potential PknG binders. A close comparison to the M. tuberculosis proteome provided a subset of 20 proteins that may interact with PknG. Our results confirmed five previously reported PknG-associated proteins: PknG, DnaK chaperone, ABC transporter Rv1747, Ribosomal Protein L23 and Elongation Factor Tu, highlighting our platform’s validity to uncover the PknG interactome. Altogether, our results reveal putative protein-protein interactions that may play a role in mycobacterial survival, while also providing solid bases for the development of anti-tuberculosis drugs by disrupting these interactions or exploiting these lead-like peptide molecules. / Tesis

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20991号 / 医博第4337号 / 新制||医||1027(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 小川 誠司, 教授 野田 亮, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

No description available.

No description available.

No description available.