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Implication du complexe d'initiation de la traduction eIF4F dans la résistance aux inhibiteurs de la voie des MAPK / The translation initiation complex eIF4F is involved in resistance to MAPK inhibitorsMalka mahieu, Hélène 14 December 2015 (has links)
Le mélanome métastatique est un des cancers les plus agressifs avec une croissance constante du nombre de nouveaux cas par an dans le monde.Deux mutations sont principalement à l’origine de ce cancer : BRAF(V600) et NRAS(Q61). Les principaux traitements sont des thérapies ciblant BRAF lui-même ou MEK, prescrites en monothérapie ou en combinaison. La moitié des patients répondent à ce traitement mais malheureusement rechutent dans les 6 mois à 1 an après le début du traitement. Les mécanismes de résistance décrits passent par une réactivation de la voie des MAPK, de la voie PI3K/Akt/mTOR ou par une dérégulation de l’apoptose.Les deux premières voies de signalisation permettent la régulation d’un complexe : le complexe d’initiation de la traduction eIF4F. Ce complexe est composé de 3 protéines : eIF4E, une protéine qui s’associe à la coiffe 7-methyl-guanosine, eIF4A une hélicase et eIF4G une protéine échafaudage dont un des rôles est de maintenir la cohésion de ce complexe.Le complexe eIF4F étant en aval de deux voies dérégulées dans le mélanome, le but de ma thèse a été de mettre en évidence une potentielle implication de ce complexe dans la résistance aux thérapies ciblant BRAF et MEK.Pour cela, dans un premier temps, nous avons traiter des lignées cellulaires de mélanome BRAF(V600) sensibles et résistantes aux anti-BRAF et anti-MEK. Nous avons ensuite quantifié les interactions eIF4E-eIF4G, synonymes d’activation du complexe eIF4F, et les interactions eIF4E-4EBP1, synonymes d’inhibition de la traduction par une technique de Proximity Ligation Assay (PLA). Ces expériences ont permis de conclure que les inhibiteurs de BRAF et de MEK induisaient une dissociation du complexe dans les lignées sensibles, alors qu’il reste maintenu dans les lignées résistantes. Ces résultats ont été confirmés sur des tumeurs de patients répondeurs et non répondeurs à ces mêmes traitements. Par la suite, nous avons reproduits ces expériences avec un panel de lignées cellulaires mutées en NRAS(Q61) et avons observé les mêmes résultats.Nous avons donc pu mettre en évidence l’implication du complexe eIF4F dans la résistance aux thérapies ciblant la voie des MAPK dans les mélanomes mutés en BRAF et en NRAS, mais également découvert une nouvelle cible thérapeutique potentielle.La seconde partie de cette thèse a consisté au test de plusieurs inhibiteurs connus et spécifiques d’eIF4A (silvestrol, flavaglines, hippuristanol, patéamine A) ou de l’interaction eIF4E-eIF4G (4EGI1). Tous ces inhibiteurs ont sensibilisé les lignées résistantes aux thérapies ciblées mais aucun ne pouvait être potentiellement utilisable en clinique.L’utilisation d’un vecteur bicistronique dans le cadre d’un criblage de drogue a permis d’identifier 4 flavaglines qui inhibent significativement le complexe d’initiation de la traduction. L’une d’entre elles (FL3) a été testé in vivo dans un modèle de xénogreffes issues de lignées cellulaires de mélanomes résistantes. Les résultats ont montré un effet synergique de cette drogue en combinaison avec le vemurafenib (anti-BRAF) ainsi qu’une inhibition de la croissance tumorale. En conclusion, nous avons identifié une nouvelle cible thérapeutique et un nouveau biomarqueur de résistance aux thérapies ciblées dans deux contextes mutationnels de mélanome différents : BRAF et NRAS. / In BRAF(V600) or NRAS(Q61)-mutant tumours, most mechanisms of resistance to drugs that target the BRAF and/or MEK kinases rely on reactivation of the RAS–RAF–MEK–ERK mitogen-activated protein kinase (MAPK) signal transduction pathway or on activation of the alternative PI(3)K–AKT–mTOR pathway (which is ERK independent). These two pathways converge to regulate the formation of the eIF4F eukaryotic translation initiation complex. By using an in situ method to detect the eIF4E-eIF4G and eIF4E-4EBP1 interactions, we recently showed that the persistent formation of the eIF4F complex, comprising the eIF4E cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase, is associated with resistance to anti-BRAF and/or anti-MEK in BRAF(V600)-mutant cancer cell lines. We next focused on NRAS-mutant cancer cell lines and found that this complex is also involved in the resistance to anti-MEK compounds. Strikingly, inhibiting the eIF4F complex in BRAF or NRAS-mutated cell lines is able to overcome resistance and to synergize with drugs targeting BRAF or MEK kinases. As a result, eIF4F appears to be a promising therapeutic target in a BRAF or NRAS-mutation context.
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Dialkynylimidazoles as irreversible MAPK inhibitors, kinase docking site probes, and anti-cancer agentsLi, Jing, Ph. D. 15 January 2013 (has links)
This dissertation research was aimed at investigating an interesting class of 1,2-dialkynylimidazoles as: 1. irreversible p38 MAP kinase α-isoform (p38α) inhibitors; 2. p38α docking site probes; 3. anti-cancer agents.
Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles was designed as potential irreversible p38α inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity were reported. Interestingly, one of the 1-ethynyl-substituted dialkynylimidazoles is a potent (IC50 = 200 nM) and selective inhibitor of p38α. Additionally, this compound covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by this compound demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors.
Blocking docking interactions between kinase network partners is a promising alternative approach for selectively inhibiting kinases. The second project involves the identification of a new class of small molecules, covalent p38α MAP kinase docking site probes. We proposed that the mechanism may involve the addition of a thiol to the N-ethynyl group. Moreover, we demonstrated that such probes can be used fluorescently to label p38α both in vitro and in cells via azide-alkyne “Click” chemistry. This serves as the basis of an assay that can be used to identify inhibitors that specifically target the substrate docking site of p38α.
The last project was focused on evaluating a new class of 1,2-dialkynylimidazoles as anti-cancer agents. One 1,2-dialkynylimidazole analog was found to be cytotoxic against a range of human cancer lines and to induce apoptosis in the human non-small cell lung cancer cell line A549. In order to elucidate the relationship between the structural basis and role of the thermal generation of diradical or carbene intermediates, a series of dialkynylimidazoles and related N-alkynylimidazoles was prepared and their cytotoxicity was determined against A549 cell line. Although the experimentally determined activation energy is in excellent agreement with that predicated from the DFT calculation, there is no correlation between the rate of Bergman cyclization and cytotoxicity to A549 cells. An alternative mechanism was proposed involving the unexpected selective thiol addition to the N-ethynyl group of certain 1,2-dialkynylimidazoles. / text
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The Effects of Amino Acids and Mitogen Activated Protein Kinase (MAPK) Inhibitors on Fluid Secretion and Ion Transport by Isolated Malpighian Tubules of Rhodnius Prolixus and Drosophila MelanogasterHazel, Matthew 09 1900 (has links)
Insect haemolynph typically contains very high levels of free amino acids 50 1 00 times that which is normal for mammalian plasma. This study examines the modulatory effects of amino acids on fluid secretion and ion transport by isolated MTs of Rhodnius prolixus and Drosophila melanogaster.
The results show that the secretion rates of isolated Malpighian tubules of both Rhodnius and Drosophila are modulated by the presence of specific amino acids in the bathing saline. Some amino acids are stimulatory, some are inhibitory and others have little or no effect. Glutamine appears to be particularly important as a stimulant of fluid secretion. As well, secreted fluid pH and Na +concentration increase and K+ concentration decreases in response to glutamine. Amino acids do not appear to be important as metabolite. in Rhodnius tubules, nor do they act to draw significant amounts of water into the lumen by osmosis. Significant stimulation of fluid secretion can be achieved by physiological levels of particular amino acids, whereas those amino acids that inhibit fluid secretion only do so at concentrations much above those at which they occur naturally in the haemolymph. Amino acids are known to be compatible osmolytes and may be acting to maintain cell homeostasis and thus to sustain fluid secretion. The passive movement of amino acids may result in cell volume changes, and some form of osmosensor is may be coupled to activation of specific kinases to produce the observed increases in fluid secretion. The effects of several kinase inhibitors were therefore examined.
The glutamine dependent increase in MT fluid secretion is blocked by two inhibitors of the stress activated protein kinase (SAPK) pathway, SP600125 and dicumoral. Inhibitors of other kinases (PKA, PKC, PKG, PI-3, p38, ERK and MEK), did not block glutamine's effects on fluid secretion rate. Alterations in cytoskeletal structure appear not to be required because cytoskeletal disrupting agents did not block the glutamine dependent inc~ease in fluid secretion, nor was the increase dependent upon protein synthesis. Results of this study are the first to suggest a role for the SAPK pathway in the control of fluid secretion rates by insect Malpighian tubules. / Thesis / Master of Science (MS)
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