Nos hospedeiros mamíferos, os parasitas do gênero Leishmania vivem nos macrófagos se evadindo de mecanismos microbicidas dessas células, tais como a produção de óxido nítrico (NO). A produção de NO pela enzima óxido nítrico sintase induzida (iNOS) nos macrófagos requer L-arginina como substrato, o mesmo aminoácido utilizado pela arginase para produzir ornitina e uréia. Logo, a arginase pode atuar na sobrevivência de Leishmania no hospedeiro competindo com a iNOS, reduzindo a produção de NO, além de seu papel na via de poliaminas, essencial para a replicação dessas células. Com isso, o objetivo desse estudo é elucidar o papel da arginase de L. (L.) amazonensis durante o ciclo de vida do parasita, particularmente, sua função no estabelecimento e na manutenção da infecção da célula hospedeira, e como esse papel seria exercido. Nesse sentido, obtivemos soros policlonais anti-arginase, a partir da arginase recombinante de L. (L.) amazonensis purificada, e esses soros foram utilizados na imunomarcação da enzima em preparações com formas promastigotas e macrófagos infectados com amastigotas de L. (L.) amazonensis. Assim, determinamos a compartimentalização da arginase nos glicossomos tanto na forma promastigota do parasita como na forma amastigota, durante a infecção. Além disso, obtivemos diversos mutantes com a expressão de arginase modificada quanto à quantidade e localização que nos permitiram avaliar a importância da compartimentalização dessa enzima nos glicossomos. Entre esses mutantes temos: superexpressores de arginase, com e sem sinal de endereçamento para glicossomo; parasitas com um alelo de arginase nocauteado e o outro substituído pelo cassete contendo o segmento ddFKBP-ARG, que teriam a expressão de arginase regulada pelo domínio ddFKB sendo nocautes funcionais de arginase; e finalmente, também obtivemos parasitas nocaute nulo de arginase. A análise desses mutantes permitiu conclusões importantes para o conhecimento da fisiologia do parasita e sua relação com o macrófago, revelando que o papel da arginase de Leishmania parece ser muito mais complexo do que o inicialmente postulado, participando na regulação de outras vias metabólicas do próprio parasita e da célula hospedeira. Paralelamente, também determinamos que o sistema ddFKBP é funcional em L. (L.) amazonensis, e assim pode ser utilizado no estudo funcional de outras proteínas importantes para esses parasitas. / In the mammal host, Leishmania parasites live inside macrophages escaping from their microbicidal mechanisms, such as the nitric oxide (NO) production. The macrophage NO production by inducible nitric oxide synthase (iNOS) requires L-arginine as substrate, the same amino acid required by arginase to generate ornithine and urea. So, arginase may play a dual role in Leishmania survival reducing the NO by competing with iNOS, and participating in the polyamines pathway, which is essential for the cells replication. Considering this, the aim of this study is to elucidate the role of L. (L.) amazonensis arginase during the parasite life cycle, mainly its function for the establishment and maintenance of the host cell infection, besides to elucidate the way that this enzyme plays its role. With this in mind, we obtained polyclonal anti-arginase sera using purified recombinant L. (L.) amazonensis arginase, these sera were used in immunolabelling assays of L. (L.) amazonensis promastigotes and macrophages infected with L. (L.) amazonensis amastigotes. These experiments determined that arginase is compartmentalized in the glycosomes of both promastigotes and amastigotes, during infection. Besides, we obtained several mutants with altered arginase expression, modified in terms of quantity and location, which permitted us to evaluate the importance of glycosome arginase compartmentalization. Among these mutants are: overexpressors of arginase, with and without glycosomal addressing signal; parasites with one arginase allele knocked out and the other one replaced by a sequence containing the ddFKBP-ARG fusion that would allow us to regulate arginase expression, working like a functional arginase knockout; and finally, we also obtained arginase null knockouts parasites. The mutants analyses lead us to important conclusions for the knowledge of the parasite physiology and its relationship with the host macrophage, revealing that the Leishmania arginase role appears to be more complex than previously thought, playing an important role in the regulation of other metabolic pathways, of the own parasite and of the host cell. In the other hand, we also determined that the ddFKBP system is functional in L. (L.) amazonensis, and then can be used for functional studies of other important parasite´s proteins.

Nos hospedeiros mamíferos, os parasitas do gênero Leishmania vivem nos macrófagos se evadindo de mecanismos microbicidas dessas células, tais como a produção de óxido nítrico (NO). A produção de NO pela enzima óxido nítrico sintase induzida (iNOS) nos macrófagos requer L-arginina como substrato, o mesmo aminoácido utilizado pela arginase para produzir ornitina e uréia. Logo, a arginase pode atuar na sobrevivência de Leishmania no hospedeiro competindo com a iNOS, reduzindo a produção de NO, além de seu papel na via de poliaminas, essencial para a replicação dessas células. Com isso, o objetivo desse estudo é elucidar o papel da arginase de L. (L.) amazonensis durante o ciclo de vida do parasita, particularmente, sua função no estabelecimento e na manutenção da infecção da célula hospedeira, e como esse papel seria exercido. Nesse sentido, obtivemos soros policlonais anti-arginase, a partir da arginase recombinante de L. (L.) amazonensis purificada, e esses soros foram utilizados na imunomarcação da enzima em preparações com formas promastigotas e macrófagos infectados com amastigotas de L. (L.) amazonensis. Assim, determinamos a compartimentalização da arginase nos glicossomos tanto na forma promastigota do parasita como na forma amastigota, durante a infecção. Além disso, obtivemos diversos mutantes com a expressão de arginase modificada quanto à quantidade e localização que nos permitiram avaliar a importância da compartimentalização dessa enzima nos glicossomos. Entre esses mutantes temos: superexpressores de arginase, com e sem sinal de endereçamento para glicossomo; parasitas com um alelo de arginase nocauteado e o outro substituído pelo cassete contendo o segmento ddFKBP-ARG, que teriam a expressão de arginase regulada pelo domínio ddFKB sendo nocautes funcionais de arginase; e finalmente, também obtivemos parasitas nocaute nulo de arginase. A análise desses mutantes permitiu conclusões importantes para o conhecimento da fisiologia do parasita e sua relação com o macrófago, revelando que o papel da arginase de Leishmania parece ser muito mais complexo do que o inicialmente postulado, participando na regulação de outras vias metabólicas do próprio parasita e da célula hospedeira. Paralelamente, também determinamos que o sistema ddFKBP é funcional em L. (L.) amazonensis, e assim pode ser utilizado no estudo funcional de outras proteínas importantes para esses parasitas. / In the mammal host, Leishmania parasites live inside macrophages escaping from their microbicidal mechanisms, such as the nitric oxide (NO) production. The macrophage NO production by inducible nitric oxide synthase (iNOS) requires L-arginine as substrate, the same amino acid required by arginase to generate ornithine and urea. So, arginase may play a dual role in Leishmania survival reducing the NO by competing with iNOS, and participating in the polyamines pathway, which is essential for the cells replication. Considering this, the aim of this study is to elucidate the role of L. (L.) amazonensis arginase during the parasite life cycle, mainly its function for the establishment and maintenance of the host cell infection, besides to elucidate the way that this enzyme plays its role. With this in mind, we obtained polyclonal anti-arginase sera using purified recombinant L. (L.) amazonensis arginase, these sera were used in immunolabelling assays of L. (L.) amazonensis promastigotes and macrophages infected with L. (L.) amazonensis amastigotes. These experiments determined that arginase is compartmentalized in the glycosomes of both promastigotes and amastigotes, during infection. Besides, we obtained several mutants with altered arginase expression, modified in terms of quantity and location, which permitted us to evaluate the importance of glycosome arginase compartmentalization. Among these mutants are: overexpressors of arginase, with and without glycosomal addressing signal; parasites with one arginase allele knocked out and the other one replaced by a sequence containing the ddFKBP-ARG fusion that would allow us to regulate arginase expression, working like a functional arginase knockout; and finally, we also obtained arginase null knockouts parasites. The mutants analyses lead us to important conclusions for the knowledge of the parasite physiology and its relationship with the host macrophage, revealing that the Leishmania arginase role appears to be more complex than previously thought, playing an important role in the regulation of other metabolic pathways, of the own parasite and of the host cell. In the other hand, we also determined that the ddFKBP system is functional in L. (L.) amazonensis, and then can be used for functional studies of other important parasite´s proteins.

Les maladies tropicales négligées, causées par des trypanosomes telles que la leishmaniose, la trypanosomiase africaine (maladie du sommeil) et la trypanosomiase américaine (la maladie de Chagas), imposent une morbidité et un taux de mortalité graves ayant un grand impact sur les populations les plus pauvres. Les traitements contemporains de ces maladies sont limités en raison des effets secondaires toxiques et de l’émergence des souches résistantes. Dans le but de répondre au besoin croissant de traitement, l’exploration de nouveaux agents antiprotozoaires est donc plus que nécessaire. La recherche décrite dans cette thèse vise à développer une nouvelle thérapie pour traiter ces infections parasitaires en ciblant de multiples fonctions vitales pour la survie du parasite. Une série de peptides, petites molécules et combinaison de peptides et petites molécules conjugués ont été préparés et étudiés. Par exemple, les lipopeptides almiramides N-méthylés présentent une activité antiparasitaire avec un indice thérapeutique élevé en ciblant le glycosome, un organite typique des trypanosomes. Une étude de la relation structure-activité a été réalisée pour examiner l’influence de la N-méthylation et de la conformation sur l’activité contre diverses souches de Leishmania et sur la cytotoxicité. La synthèse et l’analyse biologique de vingt-cinq analogues ont démontré que les dérivés portant un acide aminé Na-méthylé aux deux extrémités présentaient une activité supérieure à celle des peptides perméthylés et une puissance relativement élevée contre les souches résistantes. Le remplacement des résidus d’acides aminés dans le peptide par un α-amino γ-lactam (Agl) et N-aminoimidazalone (Nai) réduit l’activité antiparasitaire; cependant, les amides peptidiques possédant des résidus d’Agl à la deuxième position conservaient une puissance significative dans les séries non méthylées et perméthylées. L’étude systématique des effets de la N-méthylation et de la géométrie de tour sur l’activité antiparasitaire a montré la pertinence d’un conformère étendu sur les résidus centraux et la mobilité conformationnelle par isomérisation des amides tertiaires et la géométrie des tours aux extrémités des peptides actifs. L’alcaloïde naturel anisomycine a été étudié en raison de son importance en tant qu’inhibiteur de la synthèse des protéines avec une vaste activité antiparasitaire. L’évaluation des relations structure-activité antiprotozoaires de l’anisomycine a été réalisée en modifiant les groupements fonctionnels du cycle de pyrrolidine ainsi que sa partie aromatique. L’examination de la cytotoxicité et l’activité antiprotozoaire contre diverses souches de Leishmania en comparant avec le produit naturel ont révélé une stratégie de préparation d’analogues à forte puissance et à faible toxicité pour l’hôte. L’étude préliminaire de l’activité antiprotozoaire et de la sélectivité a favorisé ii l’exploration de conjugués phénoliques de l’anisomycine. Une série d’analogues de l’anisomycine ont été synthétisés en conjuguant l’anisomycine sur des peptides almiramides et des résidus d’acides aminés qui peuvent respectivement cibler les fonctions glycosome et transporteur du parasite. La cytotoxicité et l’activité antiprotozoaire contre diverses souches de Leishmania, Trypanosoma brucei et Trypanosoma cruzi ont démontré que la conjugaison de divers acides aminés et résidues insaturées donnait des agents antiparasitaires prometteurs qui ciblaient sélectivement les différents parasites avec une cytotoxité minimale en comparaison avec celle du produit naturel. L’activité antiprotozoaire de l’anisomycine et de ses dérivés a incité à poursuivre l’exploration des relations structure-activité pour le développement d’un traitement antiparasitaire. Compte tenu de l’utilité de l’anisomycine dans cette étude, une synthèse formelle du produit naturel a été développée. En utilisant la (2S,4R)-4-hydroxyproline comme précurseur chiral peu coûteux, une voie de synthèse a été développée impliquant le couplage Negishi. L’introduction dans les analogues de la décacéoxy-anisomycine a également été réalisée par un protocole similaire. Les études de relation structure-activité des peptides almiramides et des analogues de l’anisomycine décrites dans cette thèse ont ouvert une voie prometteuse pour le développement de nouveaux agents antiparasitaires à haute puissance et à faible toxicité pour l’hôte. En ciblant plusieurs points d’intervention, les nouveaux agents offrent la promesse de ralentir les modes de résistance. En outre, la synthèse d’analogues désacétoxy-anisomycine a ouvert la porte à des intermédiaires clés pour une exploration plus approfondie des dérivés de l’anisomycine et pour améliorer la synthèse de prototypes thérapeutiques afin de relever des défis importants dans l’atténuation des maladies tropicales négligées. / Neglected Tropical Diseases (NTDs) which are caused by trypasonomatid infections, such as leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease are known to inflict serious morbidity and mortality with greatest impact on the poorest populations. Contemporary anti-trypanosomatid therapy is limited due to toxic side effects and the rise of resistant stains. A growing need has emerged for novel anti-protozoal agents. The research described in this thesis seeks to develop novel therapy to treat trypasonomatid infection by targeting multiple vital functions for parasite survival. A series of peptide, small molecule, and peptide-small molecule conjugates have been prepared and studied. For example, the almiramide N-methylated lipo-peptides exhibit anti-parasitic activity with high therapeutic index by targeting the glycosome, an organelle without a mammalian counterpart. A structure-activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on specific residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide by turn inducing α-amino γ-lactam (Agl) and N-aminoimidazalone (Nai) counterparts reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides. The natural alkaloid anisomycin was studied because of significance as a protein synthesis inhibitor with broad anti-parasitic activity. Assessment of the structure - anti-protozoan activity relationships of anisomycin was performed by modifying pyrrolidine and aromatic functional groups. Examination of cytotoxicity and antiprotozoal activity against various strains of Leishmania and comparison with the natural product revealed a strategy for preparing analogs with high potency and low host toxicity. The preliminary study of antiprotozoal activity and selectivity promoted the exploration phenolic conjugates of anisomycin. A serie of anisomycin analogs were synthesized by conjugating anisomycin onto almiramide peptides and amino acids residues which iv may respectively target the glycosome and transporter functions of the parasite. The cytotoxicity and antiprotozoal activity against various strains of Leishmania, Trypanosoma brucei and Trypanosoma cruzi demonstrated that conjugation of various amino acid and unsaturated moieties yielded promising agents that selectively targeted the different parasites with minimal cytotoxity compared to that of the natural product. The antiprotozoal activity of anisomycin and derivatives has led to a better understanding of structure-activity relationships for the development of anti-parasitic therapy. Considering the utility of anisomycin in this study, a formal synthesis of the natural product was developed. Employing (2S,4R)-4-hydroxyproline as an inexpensive chiral building block, a route was conceived featuring the Negishi coupling. Entry into deacteoxy-anisomycin analogs was also achieved by a similar protocol. The structure-activity relationship studies of almiramide peptides and anisomycin analogs described in this thesis have opened a promissing gateway for developing new anti-parasitic agents with high potency and low host toxicity. By targeting multiple points of intervention, the new agents offer promise to minimize modes of resistance. Moreover, the synthesis of deacetoxyanisomycin analogs has paved the way to key intermediates for further exploration of anisomycin derivatives and for improving the synthesis of therapeutic prototypes to tackle important challenges in mitigating Neglected Tropical Diseases.