A avaliação química das folhas de Psychotria umbellata levou a identificação de quatro alcalóides pertencente ao grupo dos indol monoterpenos glicosilados, sendo psicolatina a substância majoritária. Em estudos subseqüentes, psicolatina apresentou importantes efeitos farmacológicos provavelmente relacionados com a modulação de receptores opióides, serotonérgicos 5-HT2A/C e glutamatérgicos NMDA. Assim, considerando-se a relevância das atividades biológicas descritas para psicolatina e a necessidade do estabelecimento inequívoco das características estruturais e conformacionais de compostos farmacologicamente ativos, os objetivos principais deste trabalho foram a caracterização conformacional de psicolatina e a avaliação do efeito de tratamento agudo e sub-crônico com este composto sobre os níveis de aminoácidos excitatórios e inibitórios e regiões cerebrais de roedores. A avaliação conformacional de psicolatina permitiu a caracterização de quatro confôrmeros caracterizados como mínimos de energia: 1EaCc, 1EaCd, 1EbCc e 1EbCd. As constantes de acoplamento teóricas (3JH,H) calculadas para esses confôrmeros apresentaram boa correlação com os dados experimentais de RMN, sendo que os melhores resultados foram observados para as conformações obtidas por RM1. Não foram verificadas diferenças significativas nos níveis de aminoácidos excitatórios e inibitórios em hipocampos de camundongos e ratos submetidos a tratamento agudo com psicolatina 7,5 mg/kg. No entanto, nos córtices pré-frontais dos roedores submetidos a tratamento agudo foram verificados aumentos estatisticamente significativos nos níveis dos aminoácidos excitatórios e de glutamina. Os camundongos submetidos ao tratamento agudo apresentaram elevações de 42,46 %, 50,93 % e 35,18 % nos níveis de aspartato, glutamato e glutamina, respectivamente. Os ratos submetidos ao mesmo esquema de tratamento, por sua vez, apresentaram aumentos de 43,36 % e de 57,11% nas concentrações de glutamato e glutamina. Assim como observado para o tratamento agudo, o tratamento sub-crônico com psicolatina por um período de 18 dias não provocou alterações significativas nos níveis de aminoácidos excitatórios e inibitórios em hipocampos de camundongos. Em córtices préfrontais, porém, os animais tratados com psicolatina apresentaram diminuição de 28,86 % nos níveis de glutamina. O presente estudo, corroborando dados anteriores sugere que psicolatina administrada por via intraperitoneal apresenta ação sobre o sistema nervoso central, uma vez que provocou alterações no perfil de aminoácidos excitatórios em córtex pré-frontal. / Phytochemical analyses of Psychotria umbellata leaves identified the presence of four monoterpene indole alkaloids, being psychollatine the main compound. In subsequent pharmacological investigations, psychollatine showed some important biological activities, including analgesic, anxiolytic, antidepressive and amnesic effects in mice models. These data indicate that this compound is able to modulate different neurotransmitter systems, including opioid, 5-HT2A/C and NMDA receptors. This way, considering the relevance of the pharmacological activities showed by psychollatine and the necessity of the unequivocally determination of the conformational and structural features of biologically actives compounds, the goals of this work were the conformational characterization of psychollatine and the evaluation of acute and chronic treatment with this compound on the excitatory and inhibitory amino acids levels in hippocampus and prefrontal cortex of rodents. From the conformational analyses, four minimum energy conformations were evaluates for psychollatine: 1EaCc, 1EaCd, 1EbCc e 1EbCd. The theoretical coupling constants calculated for all these conformers (3JH,H) presented a good agreement with the experimental data, mainly for the conformations obtained by RM1. No significant differences were observed on the excitatory and inhibitory amino acids levels in hippocampus of mice and rats submitted to acute treatment with psychollatine 7.5 mg/kg. Nevertheless, in prefrontal cortex of rodents submitted to acute treatment with this compound were observed increases on the excitatory amino acids and glutamine levels. Mice submitted to acute treatment with these alkaloid presented increases of 42.46 %, 50.93 % and 35.18 % on the aspartate, glutamate and glutamine levels, respectively, and rats submitted to acute treatment presented increases of 43.36 % and 57.11% on the glutamate and glutamine levels, respectively. Such as verified for the acute treatment, the chronic treatment with psychollatine 7.5 mg/kg, during 18 days, did not cause significant alterations on the excitatory and inhibitory amino acids levels in mice hippocampus. However, significant decrease on the glutamine levels (28.86 %) was observed in the prefrontal cortex of mice treated with this alkaloid during 18 days. The present work, supporting previous data, suggests that psychollatine presents effects on the central nervous system (CNS), since this compound caused alterations on the amino acids levels on prefrontal cortex.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-17T14:43:51Z No. of bitstreams: 1 PATRÍCIA PEREIRA FARIAS.PDF: 3725473 bytes, checksum: 27a790c0c992ee9cf80615ba7199bc05 (MD5) / Made available in DSpace on 2018-01-17T14:43:51Z (GMT). No. of bitstreams: 1 PATRÍCIA PEREIRA FARIAS.PDF: 3725473 bytes, checksum: 27a790c0c992ee9cf80615ba7199bc05 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As doenças parasitárias são um grave problema de saúde pública em diversos países e estão distribuídas, principalmente, em áreas endêmicas em países da África, Ásia, América Central e do Sul. Entre estas doenças estão a doença de Chagas e a doença do Sono, reconhecidas como negligenciadas. Há uma necessidade de tratamentos mais eficientes para essas doenças devido a toxicidade, baixa eficácia e segurança dos fármacos existentes, além da dificuldade de administração e evolução de resistência. O grupo de pesquisa da Dra. Núbia Boechat (Farmanguinhos/FIOCRUZ), vem realizando estudos com análogos nitroimidazólicos sintetizados considerando o megazol como protótipo, molécula ativa contra Trypanosoma cruzi, porém com efeitos mutagênicos e genotóxicos. Estes derivados apresentaram atividade contra o T. cruzi, com menor efeito genotóxico quando comparados com o megazol. Através deste trabalho, a relação estrutura-atividade dos derivados nitroimidazólicos (40a, 40b e 41a-41h) foi realizada e através dos descritores eletrônicos HOMO e LUMO, observou-se que grupos volumosos e com caráter retirador de elétrons do anel nitroimidazólico apresentam relação direta com a atividade. A avaliação do perfil toxicológico in silico confirmou que o composto 41a, mais ativo da série, não apresentou citotoxicidade em células sanguíneas humanas in vitro. O modelo da enzima nitrorredutase de T. cruzi, construído por modelagem comparativa, pode ser utilizado nos estudos de docagem molecular, os quais sugeriram que o tamanho da molécula, a possibilidade de interação com os resíduos His503 e Tyr545 e interações hidrofóbicas do tipo π-π com o cofator FMN podem contribuir para a atividade de derivados nitroimidazólicos no sítio ativo da enzima nitrorredutase. Através dos estudos de docagem molecular, sete novos derivados otimizados foram propostos (PR01 a PR07), dentre os quais o PR03, considerado como melhor ligante planejado, apresentou interações no sítio ativo similares às observadas para o protótipo 41a. Desta forma, os resultados obtidos neste trabalho podem ser úteis a novas pesquisas e podem contribuir para o desenvolvimento de novos protótipos contra o T. cruzi / Parasitic diseases are a major public health problem in many countries, and they are distributed primarily in endemic areas in Africa, Asia, Central and South America. Among them, there are Chagas disease and African trypanosomiasis, known as neglected. There is a need for better treatments for these diseases due to toxicity, low efficacy and safety of the existing drugs, besides the difficulty of administration and evolution of resistance. The research group of Dr. Núbia Boechat (Farmanguinhos/FIOCRUZ), has been conducting studies with nitroimidazole analogs synthesized through the prototype megazol (active molecule against trypanosoma, but with mutagenic and genotoxic effects). In this work, the structure activity relationship of the nitroimidazole derivatives (40a, 40b and 41a-41h) was performed and it was observed through the electronic descriptors HOMO and LUMO that groups with electron withdrawing character display relation with activity. In silico toxicological studies confirmed that the most active compound 41a did not show cytotoxicity in human blood cells in vitro. T. cruzi type I nitroreductase constructed by comparative modeling, can be used in molecular docking studies, which suggested that the size of the molecule, the possibility of interaction with the residues His503 and Tyr545, and hydrophobic interactions of the π- Π with the FMN cofactor may contribute to the activity of nitroimidazole derivatives in the active site of the nitroreductase. From molecular docking studies, seven new optimized derivatives were proposed (PR01 to PR07), among them PR03 was considered as the best planned molecule, it displayed similar active site interactions to those observed for prototype 41a. Thus, the results obtained in this work may be useful to new research and may contribute to the development of new prototypes against T. cruzi

A doença de Chagas é uma parasitose extremamente negligenciada, cujo agente etiológico é o protozoário Trypanosoma cruzi. Atualmente, 21 países da América Latina são considerados regiões endêmicas, onde 75-90 milhões de pessoas estão expostas à infecção, 6-7 milhões estão infectadas e mais de 41 mil novos casos surgem por ano. Entretanto, apenas os fármacos nifurtimox e benznidazol estão disponíveis no mercado. Estes, além da baixa eficácia na fase crônica da parasitose, apresentam diversos efeitos adversos, sendo que no Brasil apenas o benznidazol é utilizado. Este fato mostra a importância de se ampliar o número de fármacos disponíveis e propor quimioterapia mais eficaz para o tratamento da doença de Chagas. Como forma de contribuir para essa busca, este trabalho objetiva a síntese de compostos híbridos bioisostéricos N-acilidrazônicos e sulfonilidrazônicos, contendo grupo liberador de óxido nítrico, com potencial de interação com cisteíno-proteases parasitárias, tais como a cruzaína. Nestes derivados, os grupos liberadores de óxido nítrico utilizados foram os grupos furoxano (contendo substituinte metílico e fenílico) e éster nitrato. Propôs-se a variação de anéis aromáticos substituídos e não-substituídos, com o intuito de avaliar a possível relação estrutura-atividade (REA) desses análogos. Até o momento, somente os compostos da série N-acilidrazônica tiveram avaliação biológica realizada. Os valores de IC50 dos compostos na forma amastigota do parasita variaram entre >100 a 2,88 µM, sendo este último valor comparável ao fármaco de referência. A atividade inibitória frente à cruzaína foi de 25,2 µM a 2,2 µM. Já a liberação de óxido nítrico foi avaliada pelo método indireto de detecção de nitrato e os valores variaram entre 52,0 µM e 4.232,0 µM. Estes são bem inferiores ao composto padrão, além de não se identificar correlação direta entre a atividade biológica e a liberação de NO. Na sequência, os dois compostos mais ativos (6 e 14) foram submetidos a estudos de permeabilidade e de citotoxicidade. O composto 6 foi considerado o de maior permeabilidade segundo o Sistema de Classificação Biofarmacêutica (SCB) e todos os compostos apresentaram a taxa de fluxo menor que 2, indicando a ausência de mecanismo de efluxo. Na avaliação do potencial citotóxico desses compostos em células humanas, o derivado 6 apresentou índice de seletividade superior ao do benznidazol. Em estudos de modelagem molecular usando análise exploratória de dados (HCA e PCA), propriedades estéricas/geométricas e eletrônicas foram consideradas as mais relevantes para a atividade biológica. Além disso, estudos de docking mostraram que a posição do grupo nitro no anel aromático é importante para a interação com a cruzaína. Ademais o composto 6 não provocou mudanças significativas no ciclo celular e na fragmentação de DNA em células humanas, mostrando-se como líder promissor para futuros estudos in vivo. Atividade tripanomicida, citotoxicidade, potencial de liberação de NO e estudos de permeabilidade dos 23 derivados sulfonilidrazônicos e ésteres nitrato estão sendo avaliados. / Chagas disease is an extremely neglected parasitic disease whose etiologic agent is the protozoan Trypanosoma cruzi. Currently 21 Latin American countries are considered endemic regions, where 75-90 million people are exposed to infection, 6-7 million are infected and more than 41,000 new cases occur annually. However only nifurtimox and benznidazole are available on the market. These drugs, besides low efficacy in the chronic phase of the parasite have numerous adverse effects, and in Brazil only benznidazole is used. This fact shows the importance of increasing the number of drugs available and propose more effective chemotherapy for the Chagas disease treatment. As a contribution to the problem, this study aims the synthesis of biososteric compounds from N-acylhydrazone and sulfonylhydrazone, which have the potential to interact with parasitic cysteine protease, such as cruzain, containing nitric oxide releasing groups, which also has inhibitory activity in this enzyme class. In these derivatives nitric oxide releasing groups used were furoxan (containing methyl and phenyl substituent) and nitrate ester groups. The variation of aromatic rings substituted and unsubstituted was proposed in order to evaluate the possible structure-activity relationship (SAR) of these analogs. Only N-acylhydrazone series had its biological profile evaluated up to now. The IC50 values of the compounds against the amastigote form of the parasite ranged from >100 µM to 2.88 µM, the last value being comparable to that of reference drug. Cruzain inhibitory activity ranged from 25.2 µM to 2.2 µM. The nitric oxide releasing potential was evaluated using the indirect method of detection and nitrate values ranged between 52.0 µM and 4,232.0 µM. These results are below than those of the standard compound, and there is no direct correlation between the biological activity and nitric oxide releasing potential as well. Further, the two most active compounds (6 and 14) were submitted to permeability and cytotoxicity studies. Compound 6 showed the highest permeability value according to Biopharmaceutics Classification System (BCS), and both compounds showed flow rate lower than 2, indicating no efflux mechanism. In the cytotoxicity studies of these compounds in human cells, the derivative 6 showed selectivity index greater than benznidazole. In molecular modeling studies using exploratory data analysis (HCA and PCA) steric/geometric and electronic properties were considered the most relevant for biological activity. In addition, docking studies were performed and showed that the position of the nitro group on the aromatic ring is important for the interaction with cruzain. Compound 6 did not cause significant changes in cell cycle and DNA fragmentation in human cells, showing to be a promising lead compound for future in vivo studies. Trypanocidal activity, cytotoxicity assay, NO releasing potential and permeability studies of the 23 sulfonylhydrazones and nitrate ester derivatives are being evaluated.

La thèse porte sur l’étude des 2 premières enzymes de la voie du MEP: la DXS et DXR. La voie du MEP conduit à la biosynthèse des isoprénoïdes chez la plupart des bactéries, dont des pathogènes. Etant absente chez l’homme, les enzymes de cette voie cible idéale pour la recherche de nouveaux antimicrobiens. L’objectif principal était d’améliorer le développement de nouveaux antimicrobiens. Nous avons utilisé des outils computationnels : méthodes de docking et de mécanique moléculaire couplée à la méthode MM/PBSA. Nous avons identifié les résidus contribuant significativement à la fixation d’un ligand dans le site actif de la DXR. Ces résultats ont été utilisés lors de la conception de nouveaux candidats inhibiteurs de type bisubstrat, biligand et difluoro phosphonate, dont 2 ont pu être synthétisés. Nous avons également développé une méthode de synthèse donnant accès au L- et D-GAP énantiomeriquement purs, dans le but d’étudier l’énantiospécificité de la DXS face à son substrat D-GAP. / This thesis concerns the study of the 2 first enzymes of the MEP pathway: DXS and DXR. The MEP pathway permits the biosynthesis of isoprénoïdes in most bacteria, including pathogenic one. As it is not present in human, enzymes of MEP pathway are effective targets in the research of new antimicrobial drugs. The objective was to advance the development of new antimicrobiotic compounds. We used computational tools: molecular docking and molecular dynamics simulations coupled with an MM/PBSA approach. We were able to identify residues that contribute significantly to the ligand binding in the DXR active site. These results were used to guide the conception of new inhibitor models, such as bisubstrates, biligands and α,α-difluoro phosphonates, two of which were synthetized. We also developed a synthesis method to obtain L- and D-GAP as enantiomerically pure molecules. The goal was to study the enantiospecificity of DXS to its substrate, D-GAP.

Pontine et Reptine constituent de nouvelles cibles thérapeutiques encore très méconnues à ce jour. Outre leur activité ATPase, les complexes multimériques de Pontine et Reptine ont été décrits comme des hélicases capables d’ouvrir les acides nucléiques. La modélisation moléculaire constitue un outil puissant pour l’étude des systèmes protéiques et c’est pourquoi une approche par docking et dynamique a été envisagée. Au vue de la taille d’un complexe à douze sous-unités, les simulations prenant en compte tous les atomes se sont avérées trop coûteuses en termes de puissance de calcul. Une approche mésoscopique,appelée gros-grains, a donc été utilisée pour réduire le nombre de particules à traiter. Legain de temps de calcul offert par ce modèle nous a permis d’étudier les complexes de Pontine et Reptine en présence de partenaires de type ligands, l’ATP et l’ADP, et de type acide nucléique. Par le biais d’un retour au niveau atomique, une ouverture de la double hélice d’ADN a pu être observée ainsi qu’une orientation préférentielle des brins. Des hypothèses mécanistiques de l'activité hélicase du complexe ont alors pu être formulées sur la base de ces résultats. / Pontin/Reptin complexes offer new therapeutic opportunities despite the fact they are still notwell known. In addition to their ATPase activity, multimeric complexes of Pontin/Reptin were reported as hélicases able to unwind nucleic acids. Molecular modeling techniques are a powerful tool to study proteins, both a docking and molecular dynamics were applied.Considering the size of a twelve sub-units complex, simulations taking into account all atoms were too expensive in terms of computational costs. A mesoscopic approach, called coarse grain,was used to reduce the number of particles. The calculation time saved with this model allowed the study of Pontin/Reptin complexes in the presence of diverse partners like small ligands (ATP or ADP) and/or nucleic acids. Reverse transformation from coarse-grain to the atomic level led to a DNA double helix opening along to the single strands rearrangement.Several mechanistic hypotheses for the complex helicase activity were formulated from these results.

Ce travail a pour objectif d'étudier les perfomances et la régiosélectivité de deux lipases lors de l'acylation de flavonoïdes en milieu organique. Cette étude a permis de montrer que la solubilité dans l'acétonitrile, l'acétone et le tert-amyl alcool dépend de la nature du flavonoïde. La solubilité la plus élevée a été obtenue avec la naringénine et l'hepéritine et la plus faible pour la rutine et l'isoquercitrine. Les propriétés thermophysiques sont également affectées par la nature du flavonoïde. Ainsi, les flavonoïdes glycosylés possèdent un point de fusion moins élevé et une enthalpie de fusion plus élevée que ceux des aglycones. Du point de vue cinétique d'acylation, des rendements de conversion de 99% ont été obtenus avec la quercétine. Ces rendements varient en fonction de la nature du flavonoïde et du donneur d'acyle, du rapport molaire (vinyle acétate/ flavonoïde) et de la nature du solvant. Le rendement le plus faible a été obtenu avec l'hespéritine. La modélisation moléculaire de flavonoïdes dans le vide et dans des solvants a permis d'étudier le rôle de la conformation sur la solubilité et de dégager des relations structure-activité pour un certain nombre de descripteurs moléculaires. Enfin des modèles OPLS tout atomes ont été construits pour étudier par dynamique moléculaire la quercétine dans des phases condensées de solvants organiques / This work aims to study the performances and the regioselcetivity of two lipases throughout the acylation of flavonoids in organic medium. This study showed that the solubility in acetonitrile, acetone and tert-amyl alcohol depends on the nature of the flavonoid. The highest solubility, in actonitrile, was obtained with the naringenin and hesperitin and the lowest with rutin and isoquercitrin. The thermophysical properties are also affected by the nature of flavonoids. Thus glycosylated flavonoids are characterized by a low melting point and a high enthalpy of fusion compared to the aglycon ones. From the kinetic acylation data, the highest conversion yields of 99% were obtained with quercetin. These conversion yields vary according to the nature of the flavonoid and the acyl donor, the molar ratio (vinyl acetate/flavonoid) and the nature of the solvent. The lowest conversion yield was obtained with hesperitin. Molecular modeling of flavonoids in vacuum and solvents allows to study the role of conformation structure on solubility and to release the structure-activity relationship with many electronic descriptors. Finally, OPLS all atoms were built to study, by molecular dynamics, quercetin in condensed phases of organic solvents.

Nous avons développé la base HBVdb (http://hbvdb.ibcp.fr) pour permettre aux chercheurs d'étudier les caractéristiques génétiques et la variabilité des séquences du virus de l'hépatite B (VHB), ainsi que la résistance virale aux traitements. HBVdb contient une collection de séquences annotées automatiquement sur la base de génomes de référence annotés manuellement, ce qui assure une nomenclature normalisée pour toutes les entrées de la base. HBVdb est accessible via un site Web dédié avec des outils d'analyses génériques et spécialisés (annotation, génotypage, détection de profils de résistance), et des jeux de données pré-calculés. La polymérase du VHB est la principale cible des traitements anti-VHB. Les analogues de nucléos(t)ides (NA) inhibent l'activité de la transcriptase inverse (RT), mais il existe des mutations de résistance aux NA. Cependant, un autre domaine enzymatique pourrait être une cible potentielle : la RNase H, liée au domaine RT, permettant la dégradation de l'ARN durant la transcription inverse. Pour pallier l'absence d'une structure expérimentale résolue, et grâce à l'analyse de séquences à partir de HBVdb, nous avons construit le modèle par homologie de la RNase H, qui a permis de définir les caractéristiques de cette RNase H de type 1. Enfin pour vérifier des hypothèses émises à partir de ce modèle, et pour le placer dans son contexte, nous avons construit un modèle plus étendu de la polymérase du VHB, qui comprend la les domaines RT et RNase H, et contribue à répondre à la question sur l'existence d'un domaine de connexion les reliant. Nous avons utilisé notre modèle pour analyser les interactions entre le site catalytique de la RT et le ténofovir / We developed HBVdb (http://hbvdb.ibcp.fr) to allow researchers to investigate the geneticcharacteristics and variability of the HBV sequences and viral resistance to treatment. HBVdb contains a collection of computer-annotated sequences based on manually annotated reference genomes. The automatic annotation procedure ensures standardized nomenclature for all HBV entries across the database. HBVdb is accessible through a dedicated website integrating generic and specialized analysis tools (annotation, genotyping, resistance profile detection), and pre- computed datasets. The HBV polymerase is the main target of anti-HBV drugs, nucleos(t)ides analogues (NA), which inhibit the activity of reverse transcriptase (RT), but NA resistance mutations appeared. Nevertheless, another enzymatic domain could be a potential drug target: RNase H domain, linked to RT, and involved in degradation of the RNA during the reverse transcription. To overcome the lack of experimental solved structure, thanks to sequences analysis from HBVdb, we built an homology model of RNase H, which helped to define the features of this type 1 RNase H. Finally, to confirm assumptions from this model and to put it in a more global context, we built an extensive HBV polymerase model, which includes the RT and RNase H domains, and helps to answer the question about the existence of connection domain linking them. We performed analyses on this model, regarding the interactions between the RT catalytic site and the Tenofovir, mapping known resistance mutations and the most variables positions of the HBV polymerase

Les interactions protéine-protéine (IPPs) contrôlent de nombreux processus physiologiques importantsdans les cellules humaines. Une caractéristique des cancers est l'échappement des cellules àl'apoptose, qui est souvent associé à la surexpression de protéines anti-apoptotiques, membres de lafamille de protéines Bcl-2. Cette famille comprend des membres anti-apoptotiques (Bcl-2, Bcl-xL,Mcl-1) et pro-apoptotiques. Dans de nombreux cancers dont les cancers de l’ovaire chimiorésistants,l'équilibre entre les membres pro- et anti-apoptotiques de la famille de protéines Bcl-2 est altéré etconduit à la survie des cellules cancéreuses. Une des stratégies envisagées pour surmonter cettechimiorésistance est rétablir l’apoptose par l’inhibition concomitante des protéines Mcl-1 et Bcl-xL.L’objectif est de concevoir des inhibiteurs à dualité d’action visant les protéines Mcl-1 et Bcl-xL.Les travaux antérieurs du laboratoire ont permis la découverte d’un inhibiteur sélectif de la protéineMcl-1, appelé Pyridoclax. Par une approche combinant les méthodes de Fragment-Based Drug Designet Structure-Based Drug Design, à partir de la structure du Pyridoclax, la conception de dual inhibiteurs,leur synthèse et leur évaluation biologique, sont rapportées dans cette thèse. L’exploration de nouveauxespaces chimiques et biologiques est ainsi rendue possible par la mise en oeuvre de cette approche ausein de laquelle le développement de nouvelles méthodologies de synthèse dans le but de concevoirdes fragments tridimensionnels originaux sera présenté.Ces travaux de thèse ont permis de concevoir, synthétiser et caractériser plus de 90 molécules.Certaines ont montré une activité pro-apoptotique intéressante en inhibant les protéines Mcl-1 et Bcl-xLnotamment. / Protein-protein interactions (PPIs) control many important physiological processes within human cells.A hallmark of cancers is the escape of cells from apoptosis, which is often associated with theoverexpression of the anti-apoptotic proteins of the Bcl-2 family. This family comprises pro-survival(Bcl-2, Bcl-xL, Mcl-1) and pro-apoptotic members. In many cancers and, in particular, chemoresistantovarian cancers, the balance between the pro- and anti-apoptotic Bcl-2 family members is alteredleading to the survival of cancerous cells. One of the strategies used to overcome chemoresistance isto re-establish apoptosis by the concomitant inhibition of Mcl-1 and Bcl-xL proteins. Therefore, theobjective is to design dual Mcl-1/Bcl-xL inhibitors.Our groups previous work allowed the discovery of a selective Mcl-1 inhibitor, named Pyridoclax. UsingFragment-Based Drug Design and Structure-Based Drug Design approaches, from the structure ofPyridoclax, the design, synthesis and biological evaluation of dual inhibitors are reported in this thesis.The exploration of novel chemical and biological space is possible by the implementation of thisapproach. The development of synthetic methodologies for the design of new 3-dimensional fragmentswill be presented.In this work, around 90 molecules were synthesized using an approach which combined Fragment-Based Drug Design and Structure-Based Drug Design methods. Some showed a pro-apoptotic activityby inhibiting Mcl-1 and Bcl-xL proteins.

Le virus de l'hépatite C (HCV) est un virus à ARN qui synthétise ses nouveaux génomes dans les cellules hôtes infectées grâce à une ARN polymérase ARN dépendante (RdRp), appelée NS5B. Cette polymérase a été pendant longtemps une cible majeure dans la recherche d'antiviraux contre l'hépatite C. Aujourd'hui, de nombreux antiviraux ont été approuvés dans le traitement de l’hépatite C ciblant différentes protéines virales, entre autre, NS5B. Le sofosbuvir qui cible le site actif de NS5B est un antiviral qui a démontré une efficacité extraordinaire dans le traitement anti-HCV.Durant ces dernières années, les recherches sur NS5B ont permis de caractériser cette protéine, à la fois structuralement et biochimiquement. La richesse des connaissances ainsi accumulées fait de NS5B un excellent modèle pour les RdRp des virus à ARN.Toutefois, peu d'études portent sur le mécanisme d’acheminement et de sélection des ribonucléotides au site actif de NS5B, et de façon générale, sur la description atomique du mécanisme de réplication assuré par NS5B, qui implique deux dications magnésium pour la catalyse comme chez toutes les polymérases de cette famille.Durant ce travail, nous avons exploité les données structurales issues de complexes ternaires (NS5B+RNA+nucleotide) obtenus en 2015 par cristallographie à l'échelle atomique. Nous avons utilisé ces structures pour mener des études de modélisation moléculaire, essentiellement par dynamique moléculaire afin d’aborder la question de l'acheminement des nucléotides vers le site actif de NS5B.Nous avons eu recours à la fois à des méthodes de dynamiques moléculaires classiques et des méthodes de dynamiques moléculaires dites biaisées telles que différentes méthodes de dynamiques moléculaires dirigées (SMD et TMD) et la dynamique moléculaire accélérée (aMD).Nos résultats indiquent que l’acheminement du nucléotide au site actif associé à un magnésium Mg(B) est contrôlé tout au long du tunnel par différents éléments de NS5B. Initialement, le nucléotide se lie à une région proche de la boucle qui surplombe le tunnel lui permettant, grâce aux interactions qu’il établit avec sa partie triphosphate, de s’orienter base en avant. Ensuite, le nucléotide atteint un point de contrôle formé essentiellement par le motif F3 (R158) et le motif F1(E143). Le nucléotide reste lié à ce site jusqu’à l’arrivée du second dication magnésium (Mg(A)) qui provoque des réarrangements structuraux, notamment au niveau du motif F3, entrainant l’avancée du nucléotide vers le site actif. Une fois ce point de contrôle passé, le nucléotide interroge alors la base du brin d’ADN matrice sans s’insérer entièrement dans le site actif.Nos simulations ont clairement établi que les dernières étapes d’entrée du nucléotide sont finement régulées par l’arrivée du second dication magnésium qui a donc un rôle de coordinateur en plus de son rôle connu dans la catalyse.Ce mécanisme d’entrée semble être spécifique aux RdRp virales et permet de comprendre pourquoi les analogues de nucléotides peuvent être aussi efficaces contre les virus à ARN. / The hepatitis C virus is an RNA virus that synthesises its new genomes in the infected host cells thanks to an RNA-dependent RNA polymerase (RdRp) termed NS5B. This polymerase has been a prime target for antiviral therapy. Numerous direct antiviral drugs are now approved in the HCV treatment and allow very high rates of treatment success. These drugs target among others the HCV NS5B RdRp with the sofosbuvir being one of the most successful drugs.Tremendous efforts have been made in the past decades to characterize NS5B, in particular structurally and biochemically. However, there is little information about the molecular mechanisms of NS5B ribonucleotides entry and selection and in general on the atomistic details of the RNA replication mechanism, although the involvement of two magnesium dications in catalysis is well established in this family of polymerases. Since 2015, structures of ternary complexes of NS5B have been resolved by crystallography offering very valuable details about the binding of nucleotides at the NS5B active site.In this work, we took advantage of these structural data to address the ribonucleotides entry and to further explore the nucleotide addition cycle in NS5B using molecular modelling and molecular dynamics simulations. We used both conventional molecular dynamics techniques and biased simulations that enhance sampling such as Steered Molecular Dynamics (SMD), Targeted Molecular Dynamics (TMD) or accelerated Molecular dynamics (aMD).Based on our modelling results, we found that the access to the active site through the nucleotides tunnel is checked by successive NS5B elements. First, the entering ribonucleotide together with an associated magnesium Mg(B) binds next to a loop that overhangs the nucleotide tunnel and interactions with its triphosphate moiety orient it base-first towards the active site. Second, the ribonucleotide encounters a checkpoint constituted by the residues of motif F3(R158) and motif F1(E143) where it is blocked until the arrival of a second magnesium ion, the Mg(A). This allowed the motif F3 to undergo small structural rearrangements leading to the advancement of the nucleotide towards the active site to interrogate the RNA template base prior to the complete nucleotide insertion into the active site.Our simulations pointed out that these dynamics are finely regulated by the second magnesium dication, thus coordinating the entry of the correct magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. This entry mechanism is specific to viral RdRps and may explain why modified ribonucleotides can be so successful as drugs against RNA viruses.

Dans le cadre de cette thèse nous nous intéressons à la découverte de nouveaux ligands originaux de la tubuline ayant une activité inhibitrice de sa polymérisation. Pour ce faire, une étude rationnelle in silico est effectuée afin d’obtenir des molécules actives in vitro sur cette protéine. Lors de cette première année de thèse nous avons mis en place en collaboration avec l’équipe de modélisation de BioCIS – CNRS (Dr. G. Bernadat et Pr. T. Ha-Duong) un criblage virtuel sur une chimiothèque de plus de 3 millions de structures chimiques présentes dans la base de données ZINC en fonction de descripteurs structuraux. Ce criblage nous a permis de faire ressortir une trentaine de molécules potentiellement actives. Nous avons déjà synthétisé un quart de ces molécules qui sont actuellement en cours d’évaluation biologique en collaboration avec l’équipe de Biochimie et Chimie structurale des substances naturelles (Dr. J. Bignon et Dr. J. Dubois) de L’Institut de Chimie et des Substances Naturelles. Pour cette année, nous allons continuer la synthèse des molécules issues de ce criblage afin de pouvoir évaluer leurs activités sur la tubuline. En fonction des résultats biologiques, nous pourrons également effectuer des pharmacomodulations afin d’améliorer l’activité d’éventuelles touches. / As part of this work we are interested in discovering new ligands original tubulin inhibitory activity having its polymerization. To do this, rational in silico study is performed to obtain the active molecules in vitro that protein. During this first year of thesis we have developed in collaboration with the modeling team BioCIS - CNRS (Dr. G. Bernadat and Prof. T. Duong Ha.) A virtual screening a chemical library of more than 3 million chemical structures in the ZINC database according to structural descriptors. This screening allowed us to bring out thirty of potentially active molecules. We have already synthesized a quarter of these molecules that are currently being biological evaluation in collaboration with the team of Biochemistry and Structural chemistry of natural substances (Dr. J. Bignon and Dr. J. Dubois) of The Institute Chemistry and Natural Products. For this year, we will continue the synthesis of molecules from this screening in order to assess their activities on tubulin. Depending on the laboratory results, we can also perform pharmacomodulations to improve any potential ligands.