Le paludisme est une maladie parasitaire tropicale menaçant les populations dans les zones tropicales et sub-tropicales, en particulier les jeunes enfants en Afrique. En raison des résistances aux médicaments antipaludiques qui se sont propagées dans le monde entier au cours des 50 dernières années, de nouveaux médicaments sont vraiment nécessaires. La plasmodione (série benzylmenadione) a été identifiée comme un médicament-candidat antipaludique puissant, agissant selon une bioactivation rédox sur les stades sanguins asexués et sexués jeunes, mais son métabolisme est inconnu. Par conséquent, afin d'identifier les structures des métabolites actifs générés par la plasmodione antipaludique, la synthèse complète de la plasmodione 13C18-enrichie a été conçue et réalisée en 10 étapes. En outre, le procédé d'extraction pour l'étude du métabolisme de la molécule a été établi à partir de globules rouges parasités traités par la plasmodione 13C18-enrichie. D'autre part, la préparation de dérivés oxétane et N-alkylaryl de plasmodione avec une solubilité potentielle améliorée a également été réalisée par substitution nucléophilie aromatique (SNAr) et réaction de couplage Buchwald-Hartwig catalysée par le palladium, respectivement. Enfin, un complexe d'or (I) phosphole, connu comme un inhibiteur irréversible et puissant de la thiorédoxine réductase séléno-dépendante humaine, a été synthétisé et son profil antiparasitaire a été étudié sur de nombreux parasites pathogènes pour l’homme, protozoaires et helminthes en cultures. / Malaria is a tropical parasitic disease threatening populations in tropical and sub-tropical areas, especially young children in Africa. Due to the drug resistance spread all over the world in the past 50 years, new drugs are urgently needed. Plasmodione (benzylmenadione series) had been identified as a potent anti-malarial early lead drug, acting through a redox bioactivation on asexual and young sexual blood stages, but its drug metabolism is unknown. Therefore, in order to identify the structures of the active drug metabolites generated from the antimalarial plasmodione, fully 13C18-enriched-plasmodione synthesis was designed and performed in 10 steps. Furthermore, the extraction method for the drug metabolism study was established from 13C18-enriched plasmodione-treated parasitized red blood cells. On the other hand, the preparation of oxetane and N-alkylaryl derivatives of plasmodione with potential improved solubility was also investigated through aromatic nucleophilic substitution (SNAr) and palladium-catalyzed Buchwald-Hartwig coupling reaction, respectively. Finally, a gold(I) phosphole complex, known as an irreversible and potent inhibitor of the human seleno-dependent thioredoxin reductase, was synthetized and its antiparasitic profile investigated against a panel of parasites, protozoans and helminthes in cultures.

Made available in DSpace on 2014-12-17T14:10:27Z (GMT). No. of bitstreams: 1 BrunoMSW_DISSERT.pdf: 2493064 bytes, checksum: 67d2a8f0addf9c48ec5976ed766a8e15 (MD5) Previous issue date: 2012-10-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Malaria is a disease of global distribution, recognized by governments around the world as a serious public health problem, affecting more than 109 countries and territories and endangering more than 3.3 billion people. The economic costs of this disease are also relevant: the African continent itself has malaria-related costs of about $ 12 billion annually. Nowadays, in addition to chloroquine, Plasmodium falciparum is resistant to many drugs used in the treatment of malaria, such as amodiaquine, mefloquine, quinine and sulfadoxine-pyrimethamine; resistance of Plasmodium vivax to treatments, although less studied, is also reported. Nature, in general, is responsible for the production of most known organic substances, and the plant kingdom is responsible for the most of the chemical diversity known and reported in the literature. Most medicinal plants commercialized in Brazil, however, are of exotic origin, which makes the search for endemic medicinal plants, besides a patent necessity, a fascinating subject of academic research and development. This study aimed to: (i) verify the antimalarial activity of ethanolic and hydroalcoholic extracts of Boerhavia paniculata Rich. And acetonic extract of Clethra scabra Pers. in Swiss albino mice infected by Plasmodium berghei NK65, (ii) observe possible combined effects between the course of infection by P. berghei NK65 and administration of these extracts in Swiss albino mice, and (iii) conduct a preliminary study of the acute toxicity of these extracts in Swiss albino mice. All extracts notable pharmacological activities - with parasite infections inhibitions ranging from 22% to 54%.These characteristics suggest that the activities are relevant, although comparatively lower than the activity displayed by the positive control group (always above 90%). The general framework of survival analysis demonstrates an overall reduction in survival times for all groups. Necroscopy has not pointed no change in color, shape, size and/or consistency in the evaluated organs - the only exception was the livers of rats submitted to treatment to hydroalcoholic extracts: these organs have been presented in a slightly congestive aspect with mass increasing roughly 28% higher than the other two groups and a p-value of 0.0365. The 250 mg/Kg ethanolic group has been pointed out by the Dunn s post test, as the only class with simultaneous inequalities (p<0.05) between positive and negative control groups. The extracts, notably ethanol extract, have, in fact, a vestigial antimalarial activity, although well below from the ones perceived to chloroquine-treated groups; nevertheless, the survival times of the animals fed with the extracts do not rise by presence of such therapy. Both the toxicopharmacological studies of the synergism between the clinical course of malaria and administration of extracts and the isolated evaluation of toxicity allow us to affirm the absence of toxicity of the extracts at the level of CNS and ANS, as well as their non-influence on food and water consumption patterns, until dosages of 500 mg/Kg. Necroscopic analysis leads us to deduct a possible hepatotoxic effect of hydroalcoholic extract at dosages of 500 mg/Kg, and an innocuous tissue activity of the ethanol extract, in the same dosage. We propose a continuation of the studies of these extracts, with protocol modifications capable of addressing more clearly and objectively their pharmacological and toxicological aspects / A mal?ria ? uma doen?a de distribui??o global, reconhecida por governos de todo o mundo como grave problema de sa?de p?blica, ocorrendo em mais de 109 pa?ses e territ?rios e pondo em risco mais de 3,3 bilh?es de pessoas. Os custos econ?micos da doen?a s?o tamb?m relevantes: apenas o continente africano tem um ?nus de cerca de US$12 bilh?es anuais. Hodiernamente, al?m da cloroquina, o Plasmodium falciparum apresenta resist?ncia aos diversos medicamentos usados na rotina, como amodiaquina, mefloquina, quinina e sulfadoxina-pirimetamina; a resist?ncia de Plasmodium vivax, apesar de menos estudada, tamb?m ? relatada. A natureza, de um modo geral, ? a respons?vel pela produ??o da maioria das subst?ncias org?nicas conhecidas, sendo o reino vegetal respons?vel pela maior parcela da diversidade qu?mica conhecida e registrada na literatura. A maioria das plantas medicinais comercializadas no Brasil, contudo, ? de origem ex?tica, o que torna a busca por plantas medicinais end?micas, al?m de uma patente necessidade, um fascinante assunto de pesquisa acad?mica e de desenvolvimento. Este trabalho teve por objetivo: (i) verificar a atividade antimal?rica dos extratos etan?lico e hidroalco?lico de Boerhavia paniculata Rich. e acet?nico de Clethra scabra Pers. em camundongos albinos Swiss infectados por Plasmodium berghei NK65; (ii) observar poss?veis efeitos combinados entre o curso da infec??o por P. berghei NK65 e a administra??o destes extratos em camundongos albinos Swiss; e (iii) realizar um estudo da toxicidade aguda destes extratos em camundongos albinos Swiss. Notam-se, em todos os extratos, atividades farmacol?gicas not?rias com inibi??es da parasitemia variando de 22% a 54% - caracter?sticas estas que sugerem atividades relevantes, apesar de comparativamente inferior ? atividade apresentada pelo grupo controle positivo (sempre superior a 90%). O quadro geral da an?lise de sobreviv?ncia demonstra uma redu??o global dos tempos de sobrevida para todos os grupos testados. A necroscopia n?o apontou, em um quadro geral, qualquer altera??o de cor, forma, tamanho e/ou consist?ncia nos ?rg?os avaliados nos estudos a ?nica exce??o recaiu sobre os f?gados dos animais submetidos ao extrato hidroalco?lico: estes se apresentaram sob um aspecto levemente congestivo, com aumento de massa cerca de 28% superior aos outros dois grupos e um p-valor de 0,0365. O grupo etan?lico 250 mg/Kg foi apontado, pelo p?s-teste de Dunn, como a ?nica classe com desigualdades simult?neas (p< 0,05) entre os grupos controles positivo e negativo. Os extratos analisados, notadamente o extrato etan?lico, apresentam, de fato, uma atividade antiplasm?dica resquicial, embora muito abaixo da percebida para grupos tratados com cloroquina; n?o obstante, os tempos de sobrevida dos animais submetidos aos tratamentos com os extratos n?o se elevam mediante a presen?a de tal terap?utica. Tanto o estudo t?xico-farmacol?gico do sinergismo entre a evolu??o cl?nica da mal?ria e a administra??o dos extratos quanto a avalia??o isolada de toxicidade nos permitem afirmar a aus?ncia de toxicidade dos extratos em n?vel de SNC e SNA, bem como a n?o influ?ncia destes nos padr?es de consumo h?drico e alimentar, at? as doses de 500 mg/Kg. A an?lise necrosc?pica nos leva ? dedu??o de um poss?vel efeito hepatot?xico do extrato hidroalco?lico, em doses de 500 mg/Kg, e uma atividade tecidual in?cua do extrato etan?lico, em mesma dosagem. Propomos uma continua??o dos estudos destes extratos, com modifica??es protocolares capazes de abordar, de forma mais clara e objetiva, seus aspectos farmacol?gicos e toxicol?gicos

Made available in DSpace on 2015-04-20T12:31:14Z (GMT). No. of bitstreams: 1 francisco.pdf: 1029822 bytes, checksum: a55ebb42d97e615744cc7460ffefa1d1 (MD5) Previous issue date: 2011-03-24 / Caferana Picrolemma sprucei Hook a plant from the Simarubaceae family with antimalarial effects well known by the Amazon region native populations is being used without any criteria by them as a medicinal tea in order to fight this disease. The plant has a taproot that could reach over 2mts depth. On chapter one, it has been researched the regrowing of the species in its own habitat, in order to evaluate the increment of vegetal material, in a given period of four years to attain technical information on cultivated stuff and a sprout production method. The selected part of plant was the stem in natural habitat, close-cut regrowing at ground-surface. The population had 75% of plants with average height of 50 cm. On following four years, after being cut to measure average height, 75 % of plants have reached maximum height between 28 to 30cm. As a main conclusion, it could be inferred that, according field observation the caferana plant needs at least the minimum of four years to recompose its growth after being cut. It has been observed a rather slow regrowth increment, because unknown factors. On chapter two, it has been used the agronomic stake technique aiming at to evaluate plant viability for sprout production. It was adopted a casual design experiment absolutely at random in factorial scheme 4 x 4 with four types of stakes ligneous, semi-ligneous, herbaceous and semi-herbaceous and four types of soil; S1 with sand 100%, S2 with 50% sand plus 50% clay, S3 with clay 100%, and S4 with composite soil 30% plus 30% clay plus 40% sand. Best kind of soil for caferana has been S4 composite 21,67%, and best stakes were: ligneous 29,16% and herbaceous 20%. On a general mood, roots were only established after one and a half year of sprout maintenance, turning rather difficult its identification, diminishing sprout development, making non-feasible the stake-technique for sprout production. On chapter three, it has been evaluated caferana plants sprouting with seeds collected at UFAM-AM. The experiment was sketched in blocks at random, with four types of soils; S1 (100% sand); S2 (50% sand plus 50% clay); S3 (100% clay) S4 (30% composite plus 30% clay plus 40% sand), during four years. At the green-house caferana has been watered with automatic sprinkling every ten minutes at 60 second - irrigating period. The largest percentage sprouting average was with substratum S4 (41.02%) followed by S3 (31.92%) and S2 (30,29%) which do not differed between them. The worst performance has been for substratum S1 (22,94%). Germination techniques in green-house, for Picrolemma spruce Hook sprout production has had a rather medium yield. It has been concluded that there is a great dependence on moisture for this species germination, already proved by best treatments (S4, S3, S2), containing (clay and humus) moisture retaining particles, which was also verified in its habitat next to Igarapes (creeks), where they develop in larger quantities. It is highly recommended to test other sprout production techniques, as in B.O.D chambers where could prove better results than in soil substrata germination. On chapter four, it has been evaluated the analysis by genetic markers conducted by three different combinations of four EcoRI-Msel starters, resulting in a total number of 443 polymorphic bands. These data has been used for the analysis of genetic divergence among populations. It was observed low-levels of genetic divergence among them. Genetic similarity at must has been of 88.8% inside C population, 39% inside D population, being similar inside P population, indicating, and lower genetic variability on C population, then influenced to endogamy crossing. Clustering populations has being displayed in the form of a dendrogram; inferring that, populations with greater Jaccard coefficient tend to have more similarity, and trees next to each other tend to be more similar among them. / Atualmente, há um grande interesse em plantas para extração de princípios ativos com atividade antimalárica, principalmente na Amazônia, onde a doença já se alastra há tempos, sem uma ação eficaz para sua erradicação. A Caferana (Picrolemma sprucei Hook. f.) é uma planta Simaroubaceae, conhecida como antimalárica por povos nativos da região Amazônica, sendo utilizada sem critérios como chá medicinal para combate à doença. A planta possui uma raiz pivotante e atinge em torno de 2 m de profundidade. No capítulo I, foi investigado o recrescimento da espécie em seu habitat, a fim de avaliar o incremento do material vegetal, num período de quatro anos, para obter informações sobre recrescimento do material selvagem e o melhor método de produção de mudas. A planta foi cortada no caule (corte rente à superfície do solo) em seu habitat natural. A população original apresentava 75% das plantas com altura média de 50 cm. Nos quatro anos seguintes, o recrescimento médio de 75% das plantas atingiu a altura de (28 a 30 cm). Concluiu-se que a planta necessita, conforme observações em campo, não menos que quatro anos para se recompor em recrescimento depois de cortada. Tendo um incremento de recrescimento lento e com grande variabilidade, devido a fatores desconhecidos. No capítulo II, utilizou-se a técnica agronômica de estaquia a fim de avaliar a viabilidade na propagação da planta nativa para produção de mudas. Adotou-se experimento em delineamento inteiramente ao acaso em esquema fatorial (4X4) com quatro tipos de estacas (lenhosa, semi-lenhosa, herbácea e semi-herbácea) e quatro tipos de substratos: (S1) com areia (100%); (S2) com 50% de areia + 50% de solo argiloso; (S3), com solo argiloso (100%); e (S4) com 30% de terra compostada + 30% de solo argiloso + 40% de areia). O melhor substrato para caferana foi o compostado S4 (21,67%), e as melhores estacas foram: lenhosa (29,16%) e herbácea (20%). De um modo geral, as raízes só apareceram após um ano e meio de manutenção da muda, tornando difícil sua identificação, diminuindo o desenvolvimento da muda e inviabilizando a técnica de estaquia para produção das mudas nativas. No capítulo III, avaliou-se a germinação de plantas de caferana com sementes coletadas na UFAM/AM. O experimento foi delineado em blocos ao acaso, com quatro tipos de substratos: S1 (100% areia); S2 (50% areia + 50% solo argiloso); S3 (100% solo argiloso) e S4 (30% composto + 30% solo argiloso + 40% areia). Na casa de vegetação regou-se com aspersão automática a cada 10 min., com período de rega de 60s. A maior percentagem média de germinação foi com o substrato S4 (41,02%) seguido de S3 (31,92%) e S2 (30,29%) que não diferiram entre si. Obtendo o pior desempenho o substrato S1 (22,94%). A técnica de germinação em casa de vegetação para produção de mudas da espécie Picrolemma sprucei, apresentou um rendimento apenas mediano. Conclui-se que há dependência de umidade para germinação da espécie, provada pela composição dos melhores tratamentos S4, S3 e S2, contendo partículas redutoras de argila e húmus, grande retentora de água, e, também verificada em seu habitat, próximas aos igarapés, se desenvolvem em maior quantidade. Recomenda-se testar outras técnicas de produção de mudas, em câmaras de BOD, onde poderá demonstrar melhores resultados do que a germinação em substratos de solo. Com relação ao experimento com marcador molecular AFLP, capítulo IV, obteve-se um índice de similaridade genética máxima em torno de 29% para a população P (ZF2), 33% para apopulação D (Ducke) e o maior índice em torno de 87% para a população C (cultivada), mostrando definitivamente a baixa variabilidade genética em populações cultivadas, possivelmente devido ao endocruzamento. A análise por marcador genético foi realizada por sete diferentes combinações de iniciadores EcoRI-MseI, resultando num total de 443 bandas polimórficas. Esses dados foram utilizados para análise da divergência genética entre populações e dentro delas. Foram detectados baixos níveis de divergência genética entre populações. O máximo de similaridade genética foi de 88,8%, dentro de população C , o de 39% dentro da população D e semelhante dentro da P (39%), indicando uma menor variabilidade genética na população C que ficou sujeita a cruzamentos endogâmicos. O agrupamento das populações, a partir do dendrograma (Apêndice I) encontrado, sugere que populações com maior coeficiente de JACCARD, tenham maior similaridade e árvores próximas tendem a ser mais similar entre si.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-02T11:30:44Z No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-12T15:51:49Z (GMT) No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) / Made available in DSpace on 2017-05-12T15:51:49Z (GMT). No. of bitstreams: 1 robertareissoares.pdf: 1821120 bytes, checksum: 37f4636d8bd6ef3623072173760d6105 (MD5) Previous issue date: 2013-02-20 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A malária permanece ao longo dos anos como emergência global em saúde pública. São aproximadamente 87 milhões de casos e 106.820 mortes ocorridas anualmente no mundo. As principais estratégias atuais no controle da doença se baseiam no diagnóstico precoce e tratamento oportuno dos casos, devendo ser considerada a resistência dos parasitos a praticamente todos os fármacos atualmente em uso, o que torna urgente a busca por novos antimaláricos. Neste contexto, análogos de 4-aminoquinolinas e 6-mercaptopurinas contendo 1,2,3-triazol ou esteroide foram sintetizados e tiveram sua atividade antimalárica investigada utilizando o teste supressivo de Peters em modelo murino de infecção por Plasmodium berghei NK65. Dentre os 11 análogos avaliados, 5 exibiram atividade antimalárica significativa, alcançando percentuais de supressão de até 81% em relação ao controle não tratado. Além da atividade antimalárica, um composto promissor não pode apresentar efeitos indesejáveis às células do hospedeiro, sendo assim foram avaliadas a citotoxicidade e atividade hemolítica dos análogos. A maioria dos compostos foram considerados seguros às células do hospedeiro mesmo nas maiores concentrações avaliadas. Portanto, estes análogos merecem ser objeto de futuras investigações visando compor novos antimaláricos. / Malaria remains over the years as a global public health emergency. There are approximately 87 million cases and 106.820 deaths worldwide each year. The main current strategies to control the disease are based on early diagnosis and appropriate treatment cases and should be considered the resistance of parasites to almost all drugs currently in use, which makes urgent the search for new antimalarials. In this context, analogs of 4-aminoquinolines and 6-mercaptopurines containing 1,2,3-triazole or steroid were synthesized and investigated for their antimalarial activity using the 4-day suppressive test described by Peters in a murine model of infection by Plasmodium berghei NK65. Among the 11 analogues evaluated, 5 exhibited significant antimalarial activity, reaching percentages of suppression up to 81% compared to untreated control. Besides antimalarial activity, a promising compound cannot have undesirable effects on host cells, thus was evaluated the cytotoxicity and hemolytic activity of analogs. Most compounds were considered safe to host cells even at the highest concentrations evaluated. So, this analogs deserve to be object of future investigations aiming compose new antimalarials.

Le complexe bc₁ de la chaîne respiratoire mitochondriale est une bonne cible thérapeutique pour traiter le paludisme car cette enzyme est essentielle au parasite. Ses deux sites actifs, Qo et Qi, formés par le cytochrome b, ne sont pas totalement conservés entre les espèces, facilitant la découverte d’inhibiteurs à affinité différentielle, ce qui est important dans le développement de médicaments. L’atovaquone est le seul antipaludique ciblant le complexe bc₁ utilisé en médecine. L’émergence de résistance rend urgente l’étude de nouveaux inhibiteurs. Les ELQs (Endochin-like Quinolones) sont une classe d’antipaludiques particulièrement prometteuse.Pour étudier la liaison des inhibiteurs dans les sites actifs et l’effet de mutations de résistance, nous utilisons la levure et des méthodes biochimiques et bio-informatiques. Dans ce travail, nous avons étudié la relation entre mutations de résistance à l’atovaquone dans le site Qo et perte de fonction. Nous avons aussi modifié le site Qo de la levure pour qu’il mime mieux le site de l’enzyme du parasite. Les résidus «Plasmodium» altèrent le fonctionnement du site, résultant en une surproduction d’ions superoxides et une perte de croissance respiratoire, qui est restaurée par la modification d’une autre sous-unité du complexe, ISP, partenaire du site Qo, suggérant que les deux sous-unités doivent s’ajuster pour un fonctionnement correct. Nous avons analysé des polymorphismes de la région Qo observés chez l’Homme et trouvé qu’ils peuvent modifier la sensibilité du complexe à l’atovaquone, ce qui pourrait avoir un impact sur les effets secondaires du traitement. Nous avons ensuite étudié le mode d’action d’ELQ-400 et montré que ce nouvel antipaludique cible les deux sites Qo et Qi, ce qui rend l’apparition de résistance peu probable. Enfin, nous avons commencé la reconstruction du site Qi de la levure pour mimer le site du parasite.Les mutants de levure avec un complexe bc₁ «Plasmodium» semblent être de bons outils pour l’étude des inhibiteurs. Leur étude a aussi permis de comprendre mieux la structure et le fonctionnement du complexe bc₁. / The bc₁ complex of the mitochondrial respiratory chain is a good therapeutic target for the treatment of malaria as the enzyme is essential for pathogen proliferation. The two catalytic sites, Qo and Qi, formed by cytochrome b, are not fully conserved between species, facilitating the development of inhibitors with differential saffinity, which is important for the development of new drugs. At present, Atovaquone is the only antimalarial drug targeting the bc₁ complex used in medicine. The emergence of resistance makes it important to find new inhibitors, and the ELQs (Endochin-like Quinolones) are promising antimalarial candidates.In order to study the inhibitor binding to the active sites and the effect of resistance mutations, we have used yeast and a combination of biochemical and bioinformatic methods. We have studied the relationship between atovaquone resistance mutations in the Qo site and loss of function. We have also modified the yeast Qo site to make it more like the parasite site. The “Plasmodium” residues in the yeast Qo site altered its activity, which resulted in the overproduction of superoxide and the loss of respiratory growth. This could be restored by the modification of another bc₁ complex subunit interacting with the Qo site, ISP, suggesting that both these subunits need to be readjusted for correct activity. We then analyzed polymorphisms of the Qo region reported in Humans and found that they could alter the enzyme sensitivity to atovaquone, which could impact the side-effects linked to atovaquone treatment. We have also studied the mode of action of ELQ-400 and showed that this new antimalarial drug targets both the Qo and Qi sites, which would make the emergence of resistance less likely. Finally, we have started the reconstruction of yeast Qi site to make it resemble the parasite site.The yeast mutants with a “Plasmodium-like” bc₁ complex could be useful tools for the study of antimalarial drugs. These analyses have also resulted in a better understanding of the structure and function of the bc₁ complex.

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Malaria, caused by the parasite Plasmodium falciparum, has a long history as a global health threat. The vector-borne disease causes millions of deaths yearly, especially in developing countries with tropical climates that facilitate transmission. Compounding the problem is the emergence of drug-resistant strains due to overuse of outdated treatments. New compounds with antiplasmodial activity are needed to be developed as effective drugs against malaria. The hypothesis for this project is that marine microorganisms have a high likelihood of yielding novel antiplasmodial chemotypes because of their high diversity, which has not yet been explored for antimalarial development. In this project, microbes harvested and fermented by the Harbor Branch Oceanographic Institute in Fort Pierce, Florida were explored as sources for antiplasmodial natural products. Using a SYBR Green I fluorescence-based assay, 1,000 microbial extracts were screened for inhibition of the multidrug-resistant Plasmodium falciparum strain Dd2. Dose-response analysis was performed on 46 fractions from isolates whose extracts demonstrated greater-than or equal to] 70% inhibition of Dd2 at 1 micro]g/mL. To evaluate cytotoxicity, the MTS cell viability assay was used to calculate IC50 of extracts from active isolates in NIH/3T3 embryonic mouse fibroblasts. Several extracts demonstrated low IC50 in Dd2 and high IC50 in 3T3, suggesting that they contain potential lead antimalarial compounds. Extracts with high selectivity indices (potent plasmodial inhibition with low mammalian toxicity) have been prioritized for dereplication, with the goal of identifying novel active components that can be developed as antimalarial drugs.

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 the most important parasitic disease in man and it kills approximately 2,000 people each day. Pregnant women are especially vulnerable to malaria with increased incidence and mortality rates. There are indications that pregnancy alters the pharmacokinetic properties of many antimalarial drugs. This is worrisome as lower drug exposures might result in lowered efficacy and lower drug exposures can also accelerate the development and spread of resistant parasites. The aim of this research was to study the pharmacokinetics and pharmacodynamics of the most commonly used drugs for the treatment of uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy using a pharmacometric approach. This thesis presents a number of important findings that increase the current knowledge of antimalarial drug pharmacology and that may have an impact in terms of drug efficacy and resistance. (1) Lower lumefantrine plasma concentrations at day 7 were evident in pregnant women compared to that in non-pregnant patients. Subsequent in-silico simulations with the final pharmacokinetic-pharmacodynamic lumefantrine/desbutyl-lumefantrine model showed a decreased treatment failure rate after a proposed extended artemether-lumefantrine treatment. (2) Dihydroartemisinin exposure (after intravenous and oral administration of artesunate) was lower during pregnancy compared to that in women 3 months post-partum (same women without malaria). Consecutive in-silico simulations with the final model showed that the underexposure of dihydroartemisinin during pregnancy could be compensated by a 25% dose increase. (3) Artemether/dihydroartemisinin exposure in pregnant women was also lower compared to literature values in non-pregnant patients. This further supports the urgent need for a study in pregnant women with a non-pregnant control group. (4) Quinine pharmacokinetics was not affected by pregnancy trimester within the study population and a study with a non-pregnant control group is needed to evaluate the absolute effects of pregnancy. (5) Finally, a data-dependent power calculation methodology using the log likelihood ratio test was successfully used for sample size calculations of mixed pharmacokinetic study designs (i.e. sparsely and densely sampled patients). Such sample size calculations can contribute to a better design of future pharmacokinetic studies. In conclusion, this thesis showed lower exposures for drugs used to treat uncomplicated Plasmodium falciparum malaria during the second and third trimester of pregnancy. More pharmacokinetic studies in pregnant women with a non-pregnant control group are urgently needed to confirm the current findings and to enable an evidence-based dose optimisation. The data-dependent power calculation methodology using the log likelihood ratio test can contribute to an effective design of these future pharmacokinetic studies.

Les travaux de thèse exposés dans ce manuscrit portent sur l'étude chimique de quatre éponges marines collectées à Madagascar : Monanchora unguiculata, Amphimedon sp., Aulospongus gardineri et Biemna laboutei. Pour les trois premières, l'extraction, l'isolement et l'identification des métabolites secondaires par différentes techniques chromatographiques (CLMP, CLHP…) et spectroscopiques (UV-visible, SMHR, RMN 1D et 2D…) ont été envisagés. Quinze métabolites secondaires de type alcaloïdes guanidiniques, dérivés de la bromotyrosine, N-acyléthanolamines et polyynes, ont été isolés de ces éponges. Huit sont de structures nouvelles. La valorisation des molécules isolées a ensuite été envisagée via l'évaluation de leurs activités biologiques. Deux d'entres elles, la fromiamycaline (M70) et la ptilomycaline F (MU7) ont montré une excellente activité antipaludique et quatre, la fromiamycaline (M70) et les ptilomycalines E (MU6), G (MU8) et H (MU9) une cytotoxicité sur cellules KB prometteuse. L'étude de la dernière éponge, Biemna laboutei, portait sur des molécules précédemment isolées au sein du LCSNSA. Les travaux entrepris comprenaient une vérification des configurations relatives de neuf alcaloïdes guanidiniques déterminées par RMN grâce à la probabilité DP4+. Les configurations relatives déterminées par RMN de sept des molécules ont ainsi pu être confirmées et trois d'entre elles, les nétamines G (E8), P (E17) et R (E19) ont fait l'objet d'une étude plus approfondie par modélisation moléculaire afin de déterminer leur configuration absolue par comparaison de leur spectre de Dichroïsme Circulaire Électronique expérimental avec un spectre calculé. / The work described in this manuscript concerns the chemical study of four sponges from Madagascar: Monanchora unguiculata, Amphimedon sp., Aulospongus gardineri and Biemna laboutei. For the first three sponges, the study was devoted to the extraction, isolation and identification of secondary metabolites by various chromatographic (MPLC, HPLC…) and spectroscopic (UV, HRMS, 1D and 2D NMR…) techniques. Fitfteen secondary metabolites including guanidine alkaloids, bromotyrosin derivated, N-acylethanolamines and polyynes were isolated from these sponges. Eight are new molecules. The biological activities of the isolated compounds were then evaluated. Two of them, fromiamycalin (M70) and ptilomycalin F (MU7) showed a very good antimalarial activity and four of them, fromiamycalin (M70) and ptilomycalins E (MU6), G (MU8) and H (MU9) a promising cytotoxicity against KB cells. The study of the last sponge, Biemna laboutei, was performed on molecules already isolated in the LCSNSA. This work included the determination of the relative configuration of nine guanidine alkaloids by using the DP4+ probability. The relative configuration of seven molecules was confirmed. Three of them, netamines G (E8), P (E17) and R (E19) were selected for a study by molecular modeling to determine their absolute configuration by comparison of their experimental Electronic Circular Dichroism spectrum with a calculated spectrum.