Introduction - Malaria is a major global health problem, with more than 500 million reported cases and at least 1 million deaths each year. The main problem with malaria control is the emerging drug resistance. Plasmodium falciparum (P. falciparum) developed widespread resistance to antimalarial drugs such as chloroquine (CQ) and mefloquine, but not to the artemisinins. The World Health Organization (WHO) recommended artemisinin combination therapy (ACT) for the treatment of uncomplicated malaria in all chloroquine resistance areas. However, P. falciparum has recently started to display resistance to these ACTs, highlighting the need for new chemotherapeutic approaches for the treatment of P. falciparum infections. Aims - The aims of this study were: (i) to design and synthesise a new series of antimalarial hybrid drugs, consisting of dihydroartemisinin (DHA) and aminoquinoline moieties bound covalently through different, very distinctive linkers; (ii) to determine the in vitro antiplasmodial activity and cytotoxicity of the synthesised series; (iii) to ascertain whether the in vitro antiplasmodial activity of the promising compounds would be carried over in vivo against Plasmodium vinckei (P. vinckei); and, (iv) to obtain an indication of the pharmacokinetic properties of this class of antimalarial drugs by performing snapshot pharmacokinetic analysis. Methods - DHA was coupled via an aminoethylether bond to various aminoquinolines to give hybrids and hybrid-dimers. CQ-susceptible (D10 and 3D7) and CQ-resistant (Dd2) strains of P. falciparum were used to determine the in vitro antiplasmodial activity. In vitro cytotoxicity was assessed using a mammalian cell-line (Chinese Hamster Ovarian, CHO). The antiproliferative activity of the hybrid-dimers was tested against three cell lines; renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62). P. vinckei-infected mice were treated with the hybrid drugs for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip) or orally (po), with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg, in order to determine their antimalarial activity. A snapshot oral and intravenous (IV) pharmacokinetic study was performed. Results - All compounds were obtained as the 10-β-isomers and were isolated as the oxalate salts. Low nanomolar in vitro antiplasmodial activities were displayed by several compounds in this series, with IC50 values ranging from 5.15 to 29.5 nM, in comparison with the values of 2.09–5.11 nM and 21.54–157.90 nM for each of DHA and CQ respectively. All compounds displayed good selectivity towards P. falciparum in vitro (selectivity index (SI) ≥ 20). Two of the hybrids, featuring non-methylated and methylated two-carbon diaminoalkyl linkers, exerted potent in vivo antimalarial activities, with ED50 values of 1.1 and 1.4 mg/kg by ip route and 12 and 16 mg/kg po, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg ip and at 50 mg/kg po for these two hybrids, whereas artesunate was able to provide a complete cure only at 30 mg/kg ip and 80 mg/kg po. Conclusions - These compounds may provide a lead into a new class of antimalarial drugs so badly needed for treatment of resistant strains. Despite shorter half-lives and moderate oral bioavailability in comparison with DHA, two of the compounds of this series were able to cure malaria in mice at very low dosages, implicating extremely active metabolites. The optimum linker length for antimalarial activity was found to be a diaminoalkyl linker consisting of two carbon atoms, either unmethylated or bearing a single methyl group. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

Introduction - Malaria is a major global health problem, with more than 500 million reported cases and at least 1 million deaths each year. The main problem with malaria control is the emerging drug resistance. Plasmodium falciparum (P. falciparum) developed widespread resistance to antimalarial drugs such as chloroquine (CQ) and mefloquine, but not to the artemisinins. The World Health Organization (WHO) recommended artemisinin combination therapy (ACT) for the treatment of uncomplicated malaria in all chloroquine resistance areas. However, P. falciparum has recently started to display resistance to these ACTs, highlighting the need for new chemotherapeutic approaches for the treatment of P. falciparum infections. Aims - The aims of this study were: (i) to design and synthesise a new series of antimalarial hybrid drugs, consisting of dihydroartemisinin (DHA) and aminoquinoline moieties bound covalently through different, very distinctive linkers; (ii) to determine the in vitro antiplasmodial activity and cytotoxicity of the synthesised series; (iii) to ascertain whether the in vitro antiplasmodial activity of the promising compounds would be carried over in vivo against Plasmodium vinckei (P. vinckei); and, (iv) to obtain an indication of the pharmacokinetic properties of this class of antimalarial drugs by performing snapshot pharmacokinetic analysis. Methods - DHA was coupled via an aminoethylether bond to various aminoquinolines to give hybrids and hybrid-dimers. CQ-susceptible (D10 and 3D7) and CQ-resistant (Dd2) strains of P. falciparum were used to determine the in vitro antiplasmodial activity. In vitro cytotoxicity was assessed using a mammalian cell-line (Chinese Hamster Ovarian, CHO). The antiproliferative activity of the hybrid-dimers was tested against three cell lines; renal adenocarcinoma (TK-10), breast adenocarcinoma (MCF-7) and melanoma (UACC-62). P. vinckei-infected mice were treated with the hybrid drugs for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip) or orally (po), with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg, in order to determine their antimalarial activity. A snapshot oral and intravenous (IV) pharmacokinetic study was performed. Results - All compounds were obtained as the 10-β-isomers and were isolated as the oxalate salts. Low nanomolar in vitro antiplasmodial activities were displayed by several compounds in this series, with IC50 values ranging from 5.15 to 29.5 nM, in comparison with the values of 2.09–5.11 nM and 21.54–157.90 nM for each of DHA and CQ respectively. All compounds displayed good selectivity towards P. falciparum in vitro (selectivity index (SI) ≥ 20). Two of the hybrids, featuring non-methylated and methylated two-carbon diaminoalkyl linkers, exerted potent in vivo antimalarial activities, with ED50 values of 1.1 and 1.4 mg/kg by ip route and 12 and 16 mg/kg po, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg ip and at 50 mg/kg po for these two hybrids, whereas artesunate was able to provide a complete cure only at 30 mg/kg ip and 80 mg/kg po. Conclusions - These compounds may provide a lead into a new class of antimalarial drugs so badly needed for treatment of resistant strains. Despite shorter half-lives and moderate oral bioavailability in comparison with DHA, two of the compounds of this series were able to cure malaria in mice at very low dosages, implicating extremely active metabolites. The optimum linker length for antimalarial activity was found to be a diaminoalkyl linker consisting of two carbon atoms, either unmethylated or bearing a single methyl group. / Thesis (PhD (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2013

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No. of bitstreams: 1 sposito_l_me_arafcf.pdf: 2393152 bytes, checksum: cd33f70c4383729bdf5dc49ce9d3375f (MD5) Previous issue date: 2017-06-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O uso de produtos naturais na terapêutica convencional vem ganhando destaque nos últimos anos com relação a atividade antimicrobiana devido à alta taxa de efeitos tóxicos e ao excessivo crescimento do número de cepas multirresistentes aos antimicrobianos convencionais empregados na prática. Entre as novas triagens terapêuticas frente às afecções bacterianas, as infecções causadas pelo Helicobacter pylori merecem atenção especial, uma vez que estão diretamente relacionadas com quadros clínicos severos, como a gastrite crônica, úlcera péptica e câncer gástrico. A Casearia sylvestris Swartz é uma planta popularmente conhecida como guaçatonga ou erva-de-bugre, utilizada na medicina popular no tratamento de transtornos gástricos que nos últimos anos vem despertando interesse na comunidade científica, por suas importantes propriedades com potencial terapêutico como atividade anti-inflamatório, antiofídico, cicatrizante, citotóxica em linhagens de células tumorais e principalmente antiulcerogênica. Neste contexto, uma excelente alternativa para o aprimoramento dos parâmetros biodisponíveis dos extratos vegetais muito utilizada na atualidade é a incorporação destes em sistemas nanotecnológicos de liberação de fármaco para que o produto ativo tenha uma melhor ação. Deste modo, o presente trabalho teve por objetivo avaliar o potencial biológico antibacteriano in vitro do extrato etanólico, extrato aquoso, fração e subfrações das folhas de Casearia sylvestris Swartz, incorporar em um sistema de liberação de fármacos os derivados mais ativos e a partir dos resultados realizar o experimento in vivo contra a cepa ATCC 43504 de H. pylori. Os extratos foram preparados por infusão e maceração. Para as análises biológicas utilizamos a técnica de microdiluição para determinar a concentração inibitória mínima e o biofilme formado. O estudo foi desenvolvido aplicando-se metodologias in vitro e in vivo para elucidar o perfil antibacteriano pretendido, obtendo-se resultados satisfatórios, deixando esse estudo mais interessante, já que a literatura mostra que a C.sylvestris tem perfil anti-ulcerogênico. A partir desses resultados pudemos concluir que os derivados de C. sylvestris apresentaram estatisticamente atividades antibacterianas tanto in vitro como in vivo, sendo que a maioria dos derivados vegetais com exceção dos extratos aquosos, fração 1 e 3 e a subfração 17, tiveram alguma atividade frente ao micro-organismo. Essa atividade provavelmente esta relacionada aos diterpenos clerodânicos que estão em abundância nos derivados da C. sylvestris. A incorporação no sistema nanoestruturado não potencializou a atividade dos derivados vegetais como esperado na maioria dos teste, isto talvez seja pelo fato de a composição dos derivados vegetais e do sistema não serem compatíveis quimicamente. Desde que a literatura mostrou a atividade antiulcerogênica dos derivados vegetais da C. sylvestris, eles podem ser utilizados tanto na cicatrização das lesões como também para a erradicação do H. pylori. / The use of natural products in conventional therapeutics has been gaining highlight in recent years relative to the antimicrobial activity due to the high rate of toxic effects and the excessive growth of the number of multiresistant strains to he conventional antimicrobials used in practice. Among the new therapeutic screenings for bacterial diseases, infections caused by Helicobacter pylori deserve special attention, since they are directly related to severe clinical conditions, such as chronic gastritis, peptic ulcer and gastric cancer. Casearia sylvestris Swartz is a plant popularly known as guaçatonga or erva-de-bugre, used in popular medicine to the treatment of gastric disorders that in recent years has gained the interest of the scientific community due to its important properties with therapeutic potential as an anti-inflammatory, antiofidic, healing, cytotoxicity against tumor cells and mainly antiulcerogenic. In this context, an excellent alternative for the improvement of the bioavailability parameters of the vegetal extracts that is used today is the incorporation of these in nanotechnological systems of drug release so that the active product has a better action. Thus, the aim of this study was to evaluate the in vitro antibacterial potential of the ethanolic extract, aqueous extract, fraction and subfractions of the leaves of Casearia sylvestris Swartz, and incorporate into a drug delivery system the most active derivatives and from these results carry out the in vivo experiment against the H. pylori ATCC strain. The extracts were prepared by infusion and maceration. For the biological analyzes we use the microdilution technique to determine the minimum inhibitory concentration and the formed biofilm. The study was developed applying in vitro and in vivo methodologies to elucidate the antibacterial profile. From the results, we can conclude that the C. sylvestris derivatives showed statistically antibacterial activities both in vitro and in vivo. Most of the vegetal derivatives with the exception of aqueous extracts, fractions 1 and 3 and subfraction 17, had some activity against to the microorganism. This activity is probably related to clerodan diterpenes that are abundant in C. sylvestris derivatives. The incorporation into the nanostructured system did not potentiate the activity of the vegetable derivatives as expected in most tests, this is perhaps due to the fact that the composition of the plant derivatives and the system are not chemically compatible. Since the literature shows an antiulcerogenic activity of C. sylvestris derivatives, they could be used both for wound healing and for H. pylori eradication.

L'ingénierie enzymatique est un outil très avantageux dans l'industrie biotechnologique. Elle permet d'adapter les enzymes à une activité ou à une condition de réaction spécifique. En outre, elle peut permettre de déchiffrer les éléments clés qui ont facilité leur modification. Bien que l'ingénierie enzymatique soit largement pratiquée, elle comporte encore plusieurs goulets d'étranglement. Certains de ces goulets d'étranglement sont techniques, comme le développement de méthodologies pour la création de banques de mutations ciblées ou la réalisation de criblages à haut débit, et d'autres sont conceptuels, comme le déchiffrage des caractéristiques clés pertinentes d'une protéine cible pour la réussite d'un projet d'ingénierie. Parmi ces défis, l'épistasie intra-génique, ou la non-additivité des effets phénotypiques des mutations, est une caractéristique qui entrave grandement la prévisibilité. L'amélioration de l'ingénierie enzymatique nécessite une approche multidisciplinaire qui inclut une meilleure compréhension des relations structure-fonction-évolution. Cette thèse vise à contribuer à l'avancement de l'ingénierie enzymatique en étudiant deux systèmes modèles. Premièrement, des variantes dynamiques de la ß-lactamase TEM-1 ont été choisies pour étudier le lien entre la dynamique des protéines et l'évolution. La ß-lactamase TEM-1 a été largement caractérisée dans la littérature, ce qui s'est traduit par des connaissances approfondies sur son mécanisme de réaction, ses caractéristiques structurelles et son évolution. Les variantes de la ß-lactamase TEM-1 utilisées comme système modèle dans cette thèse ont été largement caractérisées, montrant une dynamique accrue à l'échelle temporelle pertinente pour la catalyse (µs à ms) mais maintenant la reconnaissance du substrat. Dans cette thèse, l'évolution in vitro de ces variantes dynamiques a été réalisée par des cycles itératifs de mutagenèse et de sélection aléatoires pour permettre une exploration impartiale du paysage de ‘fitness’. Nous démontrons que la présence de ces mouvements particuliers au début de l'évolution a permis d'accéder à des voies de mutations connues. De plus, des interactions épistatiques connues ont été introduites dans les variantes dynamiques. Leur caractérisation in silico et cinétique a révélé que les mouvements supplémentaires sur l'échelle de temps de la catalyse ont permis d'accéder à des conformations conduisant à une fonction améliorée, comme dans le TEM-1 natif. Dans l'ensemble, nous démontrons que l'évolution de la b-lactamase TEM-1 vers une nouvelle fonction est compatible avec divers mouvements à l'échelle de temps µs à ms. Il reste à savoir si cela peut se traduire par d'autres enzymes ayant un potentiel biotechnologique. Deuxièmement, la lipase Cal-A, pertinente sur le plan industriel, a été choisie pour identifier les caractéristiques qui pourraient faciliter son ingénierie. La lipase Cal-A présente des caractéristiques telles que la polyvalence du substrat et une grande stabilité thermique et réactivité qui la rendent attrayante pour la modification des triglycérides ou la synthèse de molécules pertinentes dans les industries alimentaire et pharmaceutique. Contrairement à TEM-1, la plupart des études d'évolution in vitro de la lipase Cal-A ont été réalisées dans un but industriel, avec une exploration limitée de l'espace de mutation. Par conséquent, les caractéristiques qui définissent la fonction de la lipase Cal-A restent insaisissables. Dans cette thèse, nous faisons état de la mutagenèse ciblée de la lipase Cal-A, confirmant l'existence d'une région clé pour la reconnaissance du substrat. Cela a été fait en combinant une nouvelle méthodologie de création de bibliothèque basée sur l'assemblage Golden-gate avec une visualisation structurelle basée sur des scripts pour identifier et cartographier les mutations sélectionnées dans la structure 3D. La caractérisation et la déconvolution de deux des plus aptes ont révélé l'existence d'une épistasie dans l'évolution de la lipase Cal-A vers une nouvelle fonction. Dans l'ensemble, nous démontrons que l’identification d'une variété de propriétés suite à la mutagenèse ciblée peut grandement améliorer la connaissance d'une enzyme. Cette information peut être appliquée pour améliorer l'efficacité de l'ingénierie dirigée. / Enzyme engineering is a tool with great utility in the biotechnological industry. It allows to tailor enzymes to a specific activity or reaction condition. In addition, it can allow to decipher key elements that facilitated their modification. While enzyme engineering is extensively practised, it still entails several bottlenecks. Some of these bottlenecks are technical such as the development of methodologies for creating targeted mutational libraries or performing high-throughput screening and some are conceptual such as deciphering the key relevant features in a target protein for a successful engineering project. Among these challenges, intragenic epistasis, or the non-additivity of the phenotypic effects of mutations, is a feature that greatly hinders predictability. Improving enzyme engineering needs a multidisciplinary approach that includes gaining a better understanding of structure-function-evolution relations. This thesis seeks to contribute in the advancement of enzyme engineering by investigating two model systems. First, dynamic variants of TEM-1 ß-lactamase were chosen to investigate the link between protein dynamics and evolution. TEM-1 ß-lactamase has been extensively characterized in the literature, which has translated into extensive knowledge on its reaction mechanism, structural features and evolution. The variants of TEM-1 ß-lactamase used as model system in this thesis had been extensively characterized, showing increased dynamics at the timescale relevant to catalysis (µs to ms) but maintaining substrate recognition. In this thesis, in vitro evolution of these dynamic variants was done by iterative rounds of random mutagenesis and selection to allow an unbiased exploration of the fitness landscape. We demonstrate that the presence of these particular motions at the outset of evolution allowed access to known mutational pathways. In addition, known epistatic interactions were introduced in the dynamic variants. Their in silico and kinetic characterization revealed that the additional motions on the timescale of catalysis allowed access to conformations leading to enhanced function, as in native TEM-1. Overall, we demonstrate that the evolution of TEM-1 b-lactamase toward new function is compatible with diverse motions at the µs to ms timescale. Whether this can be translated to other enzymes with biotechnological potential remains to be explored. Secondly, the industrially relevant Cal-A lipase was chosen to identify features that could facilitate its engineering. Cal-A lipase presents characteristics such as substrate versatility and high thermal stability and reactivity that make it attractive for modification of triglycerides or synthesis of relevant molecules in the food and pharmaceutical industries. Contrary to TEM-1, most in vitro evolution studies of Cal-A lipase have been done towards an industrially-specified goal, with limited exploration of mutational space. As a result, features that define function in Cal-A lipase remain elusive. In this thesis, we report on focused mutagenesis of Cal-A lipase, confirming the existence of a key region for substrate recognition. This was done by combining a novel library creation methodology based on Golden-gate assembly with script-based structural visualization to identify and map the selected mutations into the 3D structure. The characterization and deconvolution of two of the fittest revealed the existence of epistasis in the evolution of Cal-A lipase towards new function. Overall, we demonstrate that mapping a variety of properties following mutagenesis targeted to specific regions can greatly improve knowledge of an enzyme that can be applied to improve the efficiency of directed engineering.