<p>According to the World Health Organization(WHO) the most efficient and cost-effective strategies in the global fight of malaria are the Indoor Residual Spraying (IRS) and the Insecticide Treated Nets ITNs). However, since the strategies include the use of synthetic insecticides, WHO reports that they sometimes meet opposition in the society. In a Global Malaria Programme report from 20061, WHO describes that concerns in the community regarding the safety of the IRS hinder its effective implementation. WHO states that the social acceptability of ITNs2 has to increase. This study aims at investigating if and where in the Ugandan society the antimalarial strategies meet opposition. The study analyzes whether authorities, non-governmental organizations and caretakers in one region in Uganda reject the antimalarial strategies recommended by WHO. The aim is further to investigate where focus should be put in order to meet the</p><p>opposition (if any) to current strategies and thus facilitate the implementation of the strategies. The methodology used is an empirical approach based on interviews with officials at authorities, representatives of non-governmental organizations (NGOs) and caretakers in the slum areas in Kawempe Division, which is an area highly exposed to malaria, in Uganda. The results show that the authorities and the NGOs in this study accept the current strategies but believe that they are not fully accepted by caretakers. Further, the authorities and the NGOs point out that current strategies, mainly IRS, meet great resistance among politicians and within the agricultural and environmental sector. Nevertheless, the majority of the caretakers in the interviews does accept the strategies and give other reasons for not having them implemented in their houses. Many of the households do not have the money neither to buy the ITNs nor to have the IRS implemented in their houses. Thus, this study implies that the opposition to the current strategies is not among authorities, NGOs or caretakers but in the political, environmental and agricultural</p><p>sphere. In order to fight malaria in the study area, WHO and stakeholders have to work with the change of attitudes among politicians and stakeholders within the environmental and agricultural sector in Uganda. They also have to provide poor households with ITNs or IRS for free, since lack of money is the reason for the studied group of caretakers not having the recommended strategies implemented in their houses.</p>

According to the World Health Organization(WHO) the most efficient and cost-effective strategies in the global fight of malaria are the Indoor Residual Spraying (IRS) and the Insecticide Treated Nets ITNs). However, since the strategies include the use of synthetic insecticides, WHO reports that they sometimes meet opposition in the society. In a Global Malaria Programme report from 20061, WHO describes that concerns in the community regarding the safety of the IRS hinder its effective implementation. WHO states that the social acceptability of ITNs2 has to increase. This study aims at investigating if and where in the Ugandan society the antimalarial strategies meet opposition. The study analyzes whether authorities, non-governmental organizations and caretakers in one region in Uganda reject the antimalarial strategies recommended by WHO. The aim is further to investigate where focus should be put in order to meet the opposition (if any) to current strategies and thus facilitate the implementation of the strategies. The methodology used is an empirical approach based on interviews with officials at authorities, representatives of non-governmental organizations (NGOs) and caretakers in the slum areas in Kawempe Division, which is an area highly exposed to malaria, in Uganda. The results show that the authorities and the NGOs in this study accept the current strategies but believe that they are not fully accepted by caretakers. Further, the authorities and the NGOs point out that current strategies, mainly IRS, meet great resistance among politicians and within the agricultural and environmental sector. Nevertheless, the majority of the caretakers in the interviews does accept the strategies and give other reasons for not having them implemented in their houses. Many of the households do not have the money neither to buy the ITNs nor to have the IRS implemented in their houses. Thus, this study implies that the opposition to the current strategies is not among authorities, NGOs or caretakers but in the political, environmental and agricultural sphere. In order to fight malaria in the study area, WHO and stakeholders have to work with the change of attitudes among politicians and stakeholders within the environmental and agricultural sector in Uganda. They also have to provide poor households with ITNs or IRS for free, since lack of money is the reason for the studied group of caretakers not having the recommended strategies implemented in their houses.

Ces travaux de thèse décrivent la synthèse de trente-quatre nouveauxdérivés de sulfanyl et de sulfonyl chalcones et de trente-quatre nouveaux dérivés dumétronidazole, ainsi que leurs évaluations biologiques en tant que possibles agentsantipaludiques et leishmanicides. L'évaluation antimalarique in vitro sur la formationde la β-hématine a montré que douze de ces dérivés possèdent une activitéinhibitrice supérieure à 80%. In vivo, deux composés ont conduit à une diminution dela parasitémie au quatrième jour après infection et à une augmentation significativedu temps de survie chez la souris. Dans le cas de l’évaluation leishmanicide in vitro,trois composés ont montré une activité inhibitrice sur la croissance despromastigotes des espèces L. mexicana et L. braziliensis. Les composés les plusactifs sont des dérivés de type benzoate possédant des substituants hydroxyles surles positions 3,4,5 et 3,4 du cycle benzénique. / This research work describes the chemical synthesis of thirty-four newsulfanyl and sulfonyl chalcone derivatives, and thirty-four new metronidazolederivatives, as well as their biological assays as antimalarial and leishmanicidalagents. The antimalarial in vitro evaluation on the β-hematin formation showed thattwelve of these derivatives display an inhibitory activity higher than 80%. In vivo, twocompounds were found to decrease the parasitaemia by the fourth day after infectionand to increase significantly the survival time of mice. In the case of the in vitroleishmanicidal evaluation, three compounds showed an inhibitory activity on thegrowth of promastigotes of L. mexicana and L. braziliensis species. The most activecompounds are benzoate derivatives featuring hydroxyl substituents at the 3,4,5 and3,4 positions of the benzene ring.

Neste trabalho foram estudadas, por espectroscopia UV-Vis, as interações do fármaco anti-malária cloroquina com uma série de porfirinas meso fenilas substituídas aniônicas, catiônicas e neutras, e seus complexos Fe(III), em tampão aquoso e em solução metanólica. Com base nos resultados de interação, as ferro(III) porfirinas meso fenilas substiuídas e a FePPIX foram utilizadas como catalisadores para a oxidação de cloroquina por iodosilbenzeno (PhIO) e peróxidos (H2O2 e ácido meta-cloroperbenzóico, CPBA), em ambos os meios. Dos estudos de interação foi observado, em meio aquoso, pH 6,4, a formação de um complexo p-p, resultante do efeito cooperativo das interações eletrostáticas e da interação p-p entre as espécies aniônicas e a cloroquina. Em metanol nenhuma complexação p-p foi observada. As porfirinas e ferro(III) porfirinas neutras e catiônicas não formaram complexos com a cloroquina. As constantes de formação do complexo p-p entre as porfirinas ou ferro(III) porfirinas e cloroquina foram comparáveis áquelas relatadas na literatura para a cloroquina e outras ferro(III) porfirinas naturais. As reações de oxidação foram analisadas por cromatografia líquida de alta eficiência (CLAE). O principal produto obtido em todos os sitemas estudados foi a monodesetilcloroquina. Este é também o principal produto ?in vivo? da oxidação deste fármaco catalisada por CYP-450. A identificação do produto que é gerado em segunda maior quantidade, o derivado desetilamino da cloroquina, foi realizada por CLAE acoplada a espectrometria de massas. Dos resultados das reações de oxidação com PhIO e H2O2 foi possível constatar que: as ferro(III) porfirinas aniônicas são os catalisadores mais eficientes, com rendimentos de até 30 % de monodesetilcloroquina; em solução aquosa elas são seletivas para a deetilação oxidativa da cloroquina, enquanto que em metanol até cinco produtos de oxidação podem ser observados. As ferro(III) porfirinas neutras apresentam baixa atividade catalítica na oxidação da cloroquina por PhIO ou H2O2 pois não interage eletrostaticamente com o substrato. As ferro(III) porfirinas catiônicas não apresentam atividade catalítica devido à repulsão eletrostática com o substrato. As reações de oxidação por CPBA apresentaram baixos rendimentos devido à rápida destruição dos catalisadores quando se utilizou este oxidante. Foram ainda realizados estudos catalíticos utlizando ferro(III) porfirinas suportadas em membrana de polidimetilssiloxano (PDMS) e poli-(1?4)-b-d-glucosamina (quitosana) na oxidação de substratos padrões (ciclocteno, cicloexano e estireno). Destes, apenas a quitosana se mostrou um suporte adequado, resultando em um catalisador eficiente. As ferro(III) porfirinas imobilizadas neste suporte foram utlizadas na oxidação da cloroquina por PhIO, levando aos mesmos rendimentos que estas ferro(III) porfirinas apresentaram em meio homogêneo, mostrando também a dependência interação(FeP:substrato)/atividade catalítica previamente observada para as ferro(III) porfirinas em solução. Os resultados obtidos neste trabalho mostram que as ferro(III) porfirinas aniônicas são eficientes catalisadores biomiméticos da oxidação da cloroquina por PhIO e H2O2, tanto em solução como imobilizados. Além disso, pode-se concluir que a habilidade do catalisador em interagir com o substrato apresenta forte influência na atividade catalítica e deve ser levada em consideração nos estudos de outros sistemas modelos do CYP-450. / In this work, the interaction between the antimalarial drug chloroquine with a series of anionic, cationic, and neutral meso phenyl substitued porphyrins; and their iron(III) complexes, was investigated by UV-Vis spectroscopy, in aqueous buffer and methanolic solution. On the basis of the interaction results, the meso phenyl substitued iron(III) porphyrins and FePPIX were used as catalyst in the oxidation of chloroquine by iodosilbenzene (PhIO) or peroxides (H2O2 e meta-chloroperbenzoic acid, CPBA), in both media. From the interaction studies, it was possible to observe the formation of a p-p complex generated from the cooperative effect of the electrostatic and p-p interactions between the anionic species and chloroquine. In methanol, no complexation was observed. The neutral and cationic porphyrins and iron(III) porphyrins did not form complexes with chloroquine. The association constants of the p-p complex formed between the meso phenyl substitued porphyrins and iron(III) porphyrins with chloroquine were comparable with those reported in the literature for the interaction of chloroquine with others natural iron(III) porphyrins. The oxidation reaction produtcts were analized by high perfomance liquid chromatography (HPLC). The major product obtained in all the studied systems was monodesethylchloroquine. This compound is also the main ?in vivo? product of chloroquine metabolism catalised by CYP-450. The identification of the second most abundant product, the desethylamine derived from chloroquine, was performed by HPLC coupled with mass spectrometry. The results obtained from the oxidation reactions of chloroquine by PhIO and H2O2 indicated that: (i) the anionic iron(III) porphyrins are efficient catalysts, with yields close to 30 % for monodesethylchloroquine; (ii) in aqueous buffer they are selective toward the oxidative N-deethylation of chloroquine, while in methanol up to five oxidation products are obtained. The neutral iron(III) porphyrins presented low catalytic activity for the chloroquine oxidation by PhIO or H2O2 due to the lack of electrostatic interaction between the catalyst and the substrate. (iii) The cationic iron(III) porphyrins did not exhibit any catalytic activity because of electrostatic repulsion tha tales place between the catalyst and the substrate. Catalyst degradation occurred when CPBA was used as oxidant, leading to very low yields of oxidation products. Catalytic studies using iron(III) porphyrins supported in polydimethylsiloxane (PDMS) membranes and in poly-(1?4)-b-d-glucosamine (chitosan) were also performed for the oxidation of standard substrates (cicloctene, ciclohexane and styrene). Only chitosan was considered to be an adequate support, resulting in efficient catalysis. The iron(III) porphyrins immobilized on this support were used for the oxidation of chloroquine by PhIO, leading to the same yields as those obtained with the same iron(III) porphyrins in homogeneous medium. It is noteworthy that the relations between the interaction FeP:substrate and the FeP catalytic activity previously observed for the iron(III) porphyrins in solution was also observed in these heterogeneous catalysis. The results obtained in this work show that anionic iron(III) porphyrins are efficient biomimetic catalysts for the oxidation of chloroquine by PhIO and H2O2, both when supported and in solution. Also, the catalyst ability to interact with the substrate has a strong influence on its activity and must be considered when studying other CYP-450 model systems.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-09-22T17:54:20Z No. of bitstreams: 1 lucaslopardifranco.pdf: 1498046 bytes, checksum: aa3cf634965e77b44f29d47f725c19c1 (MD5) / Approved for entry into archive by Diamantino Mayra (mayra.diamantino@ufjf.edu.br) on 2016-09-26T20:30:01Z (GMT) No. of bitstreams: 1 lucaslopardifranco.pdf: 1498046 bytes, checksum: aa3cf634965e77b44f29d47f725c19c1 (MD5) / Made available in DSpace on 2016-09-26T20:30:01Z (GMT). No. of bitstreams: 1 lucaslopardifranco.pdf: 1498046 bytes, checksum: aa3cf634965e77b44f29d47f725c19c1 (MD5) Previous issue date: 2010-12-20 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Este trabalho trata da síntese de diidroperóxidos e de tetraoxanos, ambos contendo um esqueleto carbônico similar àqueles das chalconas e curcuminas. Essas classes de compostos possuem um amplo espectro de ação terapêutica, o que somado ao núcleo endoperóxido pode resultar em novos candidatos a agentes antimaláricos e antiparasitários em geral. A partir de reações usando como material de partida o benzaldeído e dois de seus derivados metoxilados, foi obtida uma série de três dibenzalcetonas com estruturas similares às chalconas e curcuminas, com rendimentos que variaram de 76 a 95%. Com a finalidade de averiguar a importância da insaturação na atividade biológica dos produtos finais, foi feita a hidrogenação dessas dibenzalcetonas, gerando outras três cetonas saturadas, sendo uma delas inédita, com rendimentos que variaram de 51 a 93%. Essas seis cetonas, juntamente com a cicloexanona e a terc-butil-cicloexanona comerciais, foram usadas como materiais de partida na síntese dos diidroperóxidos e tetraoxanos. Para tal, foram testados vários métodos visando obter uma metodologia de preparação rápida, usando reagentes de baixo custo e pouco tóxicos. Nas tentativas de síntese dos diidroperóxidos foram utilizados e comparados quatro procedimentos diferentes e o mais eficiente, através do uso de peróxido de hidrogênio em fase etérea, proporcionou a obtenção de cinco diidroperóxidos, sendo três deles inéditos, com rendimentos que variaram de 51 a 89%. Nas tentativas de síntese dos tetraoxanos foram utilizados e comparados três métodos diferentes e o mais eficiente, usando a síntese “one pot”, proporcionou a obtenção de quatro tetraoxanos, sendo três deles inéditos, com rendimentos que variaram de 22 a 70%. Todos os compostos foram caracterizados através de técnicas adequadas, tais como: IV, RMN de 1H e de 13C e faixa de fusão. Esses compostos foram avaliados quanto às suas atividades tripanocidas e estão sendo avaliados quanto às suas ações antimaláricas. / This work describes the synthesis of dihydroperoxides and tetraoxanes, both with the similar structure of chalcones and curcumins. These classes of compounds have a broad spectrum of therapeutic action, which, added to the endoperoxide moiety, may result in new candidates as antimalarial and antiparasit agents. A series of three aromatic ketones were obtained from benzaldehyde and two of its methoxylated derivatives in 76 to 95% yields. These compounds contain structures similar to curcumin and chalcones. Aiming to establish the importance of unsaturation in biological activity of the final products the dibenzalketones obtained were hydrogenated, generating three more ketones in 51 to 93% yields. These six ketones, cyclohexanone and tert-butyl cyclohexanone were used in the synthesis of dihydroperoxides and tetraoxanes. For this purpose, several methods were tested aiming a fast method of preparation, using reagents of low cost and toxicity. Four different methodologies were used and compared for the synthesis of the dihydroperoxides. The most efficient provided five dihydroperoxides in 51 to 89% yields. Three different methodologies were used and compared for the synthesis of the tetraoxanes. The most efficient methodology provided four tetraoxanes in 22 to 70% yield. All compounds were characterized using appropriate techniques such as IR, 1H and 13C NMR and melting point. These compounds demonstrated low trypanocidal activity and are being evaluated for their antimalarial action.

Due to the increasing resistance against the currently used antimalarial drugs, novel chemotherapeutic agents that target new metabolic pathways for the treatment of malarial infections are urgently needed. One approach to the drug discovery process is to use interaction analysis to find proteins that are involved in a specific metabolic pathway that has been identified as a drug target. Protein-protein interactions in such a pathway can be preferential targets since a) there is often greater structural variability in protein-protein interfaces, which can lead to more effective differentiation between the parasite and host proteins; and b) the important amino acids in a protein-protein interface are often conserved and even one amino acid mutation can lead to the dissociation of the complex, implying that resistance should be slower to appear. Since polyamines and their biosynthetic enzymes occur in increased concentrations in rapidly proliferating cells, the inhibition of polyamine metabolism is a rational approach for the development of antiparasitic drugs. Polyamine synthesis in P. falciparum is uniquely facilitated by a single open reading frame that encodes both rate-limiting enzymes in the pathway, namely ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). The AdoMetDC/ODC domains are assembled in a heterotetrameric bifunctional protein complex of ~330 kDa. Inhibition of both decarboxylase activities is curative of murine malaria and indicates the viability of such strategies in malaria control. It was hypothesized that protein ligands to this enzyme can be utilized in targeting the polyamine biosynthetic pathway in a novel approach. The bifunctional PfAdoMetDC/ODC was recombinantly expressed with a C-terminal Strep-tag-II to allow affinity purification. Subsequent gel electrophoresis analysis showed the presence of 3 contaminating proteins (~60 kDa, ~70 kDa and ~112 kDa) that co-elute with the ~330 kDa AdoMetDC/ODC. Efforts to purify the bifunctional protein to homogeneity included subcloning into a double-tagged vector for tandem affinity purification as well as size-exclusion HPLC. SDS-PAGE analysis of these indicated that separation of the four proteins was not successful, implicating the presence of strong protein-protein interactions. Western blot analysis showed that the ~112 kDa and ~70 kDa peptides were recombinantly produced with a C-terminal Strep-tag, indicating their heterologous origin. The ~60 kDa fragment was however not recognised by the tag-specific antibodies. This implies that this fragment is of E. coli origin. MS-analysis of the contaminating bands showed that the ~112 kDa peptide is an N-terminally truncated form of the full-length protein, the ~70 kDa peptide is a mixture of N-terminally truncated recombinant protein and E. coli DnaK and the ~60 kDa peptide is E. coli GroEL. A P. falciparum cDNA phage display library was used to identify peptide ligands to PfAdoMetDC/ODC. Of the peptides isolated through the biopanning process, only one was shown to occur in vivo. It could however not be conclusively shown that the isolated peptides bind to PfAdoMetDC/ODC and not to the co-eluting E. coli proteins. It is thought that while it is extremely likely that interacting protein partners to PfAdoMetDC/DOC exist, the available technologies are not sufficient to lead to the identification of such partners. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2008. / Biochemistry / unrestricted

Malaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel naturallike synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have identified a novel compound, 3-amino-2-piperidinequinoline (APQ), which we termed UCF401. APQ demonstrated IC[sub50] at submicromolar concentrations against Plasmodium falciparum using the SYBR Green-I fluorescence assay measuring cellular proliferation. This compound also demonstrated low cytotoxicity against the NIH3T3 and HEPG2 cells using MTS assays, showing an IC50 of 174 [micro]M and 125 [micro]M respectively, suggesting of excellent selectivity. We evaluated the compliance of APQ with Lipinski's parameters and determined the in vitro physicochemical profiles of the compound. Our results show that APQ is a Lipinski parameter compliant and has good physicochemical properties. The cellular mechanism of action of APQ was characterized through the assessment of the effects of the compound at different stages of the parasite's intraerythrocytic life cycle. This assay was done by treating a synchronized cell line with the compound at 5X the IC50 value and then imaging the cells at 12-hour intervals. We found that APQ arrests parasite development at the trophozoite stage. In addition we determined that APQ is parasitocidal after a 96 h exposure. These results demonstrate that APQ can be considered as a validated hit and/or early lead.

As malaria, caused by Plasmodium spp., continues to afflict millions of people worldwide, there is a dire need for the discovery of novel, inexpensive antimalarial drugs. Although there are effective drugs on the market, the consistent development of drug resistant species has decreased their efficacy, further emphasizing that novel therapeutic measures are urgently needed. Natural products provide the most diverse reservoir for the discovery of unique chemical scaffolds with the potential to effectively combat malarial infections, but, due to their complex structures, they often pose extreme challenges to medicinal chemists during pharmacokinetic optimization. In our laboratory we have performed unbiased, cell-based assays of numerous synthetic compounds from chemical libraries enriched with nature-like elements. This screening has led to the discovery of many original chemical scaffolds with promising antimalarial properties. In an attempt to further characterize these scaffolds, the most promising compounds were assayed in order to determine their cytotoxic effects on mammalian cells. In addition, the development of a drug resistant parasite line of Plasmodium falciparum to the most promising compound was done in order to determine the relative probability for parasite resistance development.

Malaria, the disease caused by Plasmodium sp., claims the lives of over 1 million people every year, with Plasmodium falciparum causing the highest morbidity. Rapidly acquiring drug resistance is threatening to exhaust our antimalarial drug arsenal and already requires the utilization of combination drug therapy in most cases. The global need for novel antimalarial chemical scaffolds has never been greater. Screening of natural product libraries is known to have higher hit rates than synthetic chemical libraries. This elevated hit rate is somewhat attributed to the greater biodiversity available in natural products. Marine life is the most biodiverse system on the planet, containing 34 of the 36 known phyla of life, and is expected to be a rich source of novel chemotypes. In collaboration with the Harbor Branch Oceanographic Institute in Ft. Pierce we have screened a library of over 2,800 marine macroorganism peak fractions against Plasmodium falciparum using the SYBR green I fluorescence-based assay. In this screening process we have identified six compounds from five novel chemical scaffolds all of which have low micromolar to submicromolar IC50 values and excellent selectivity indices. Additionally, one of these chemical scaffolds, the bis(indolyl)imidazole, was selected for further in vitro pharmacological and structure-activity relationship (SAR) profiling, key steps in the challenging process of identifying a new antimalarial drug lead compound.

No / The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of ß-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg-1 day-1 ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (¿Tm value) or toxicity in the mouse¿malaria model.