Made available in DSpace on 2014-06-12T15:52:32Z (GMT). No. of bitstreams: 2 arquivo5020_1.pdf: 1527733 bytes, checksum: c4a27a4c47abc632b72b7a59590fff40 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2003 / Os mixomicetos produzem substâncias com o potencial de inibir microrganismos patógenos ao homem, como evidenciado por estudos in vitro. Estes estudos são, porém, limitados pela dificuldade em se obter material suficiente para experimentos laboratoriais. Visando contribuir para um maior conhecimento sobre o potencial biotecnológico dos mixomicetos, principalmente no que se refere à produção e identificação de substâncias bioativas, desenvolveu-se uma técnica de imobilização plasmodial, com o objetivo de produzir substâncias em quantidade que permita a realização de testes químicos e biológicos. Physarella oblonga foi selecionada para este trabalho por ser uma espécie pouco estudada do ponto de vista químico e biológico, e freqüentemente encontrada em áreas de Floresta Atlântica de Pernambuco, Brasil. Fragmentos plasmodiais, dispersos em meio de cultura líquido, foram mantidos em um biorreator, utilizando-se caulinita como matriz de enclausuramento e acetato de sódio como precursor metabólico. Eluatos plasmodiais foram coletados periodicamente e extraídos com éter/acetato de etila (65:35 v/v) e clorofórmio/acetonitrila (60:40 v/v). Os extratos orgânicos foram submetidos à leitura espectrofotométrica em 266 nm e 310 nm e à cromatografia em camada delgada, para avaliar a produção de substâncias. Testes de difusão em meio sólido foram utilizados para detectar a atividade antimicrobiana dos extratos contra bactérias Gram positivas, Gram negativas, álcool-ácido resistentes e leveduras. As substâncias bioproduzidas foram isoladas por cromatografia em sephadex LH-20 e avaliadas através de cromatografia líquida de alta eficiência e de reações de coloração com reagentes específicos, em cromatografia de camada delgada. A técnica introduzida permite que o plasmódio de P. oblonga se mantenha metabolicamente ativo quando imobilizado em caulinita, uma vezque a produção final de substâncias foi superior ao peso inicial do plasmódio imobilizado. P. oblonga tem a capacidade de produzir substâncias com atividade antimicrobiana e o processo de imobilização não altera esta capacidade; as substâncias bioproduzidas atuaram inibindo o desenvolvimento de colônias de Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa e Mycobacterium smegmatis. Notável inibição foi observada também frente às leveduras Candida albicans e Candida tropicalis. Após um ano de cultivo em câmara-úmida, o plasmódio de P. oblonga perdeu a capacidade de inibir estes mesmos microrganismos, provavelmente em conseqüência da diminuição na competição com outros organismos de seu ambiente natural. Duas frações com atividade antimicrobiana foram isoladas a partir do extrato plasmodial de P. oblonga, no qual foram detectadas substâncias pertencentes aos grupos de compostos fenólicos e esteróides

Ce travail est consacré au développement de la stratégie TDAE dans le cadre de la synthèse de nouveaux composés à visée thérapeutique. Dans une première partie, la pharmacomodulation d’un composé potentiellement ligand sélectif du récepteur 5-HT7 nous a conduit à développer une méthodologie d’addition et d’estérification séquentielle « one-pot » initiée par le TDAE. Par la suite, nous avons étendu la réactivité initiée par le TDAE aux réactions de substitution nucléophile de type 2 sur des électrophiles -halogénoesters et -halogénoamides en série nitrobenzylique. Cette méthodologie nous a permis de synthétiser différents 3-(2-nitrophényl)propanamides qui ont manifesté, pour certains, une activité analgésique significative chez la souris. La deuxième partie est consacrée à la synthèse de divers 3-phénylpropanamides et à l’évaluation de leur propriété analgésique. L’un des ces composés s’est révélé puissant mais modérément efficace par rapport à l’aspirine, que ce soit par voie injectable ou par voie orale. La troisième partie traite de la réactivité initiée par le TDAE sur le noyau 4-chloroquinazoline. Outre la mise en évidence de réactivités particulières liées aux caractères basique et nucléophile du TDAE, cette étude a montré la possibilité de réaliser des réactions de substitution nucléophile aromatique. La synthèse de diverses 4-benzyl-2-trichlorométhylquinazolines par cette méthodologie, s’inscrit dans une étude de pharmacomodulation de nouveaux agents antiplasmodiaux. / This work is focused on the development of the TDAE strategy aiming at the preparation of new bioactive compounds. In a first part, the pharmacomodulation study of a potential 5-HT7 receptor ligand led us to develop a “one-pot” methodology of addition and sequential esterification initiated by the TDAE. Later, we extended the reactivity of carbanions generated by TDAE to type 2 nucleophilic substitution (SN2) reactions on various -haloesters and -haloamides in nitrobenzylic series. This methodology allowed us to synthesize different 3-(2-nitrophenyl)propanamides which expressed a significant analgesic activity on mice. The second part is dedicated to the synthesis of various 3-phenylpropanamide and the evaluation of their analgesic potentiality. One of those compounds appeared active but moderately effective with regard to aspirin, both by infusion or oral route. The third part concerns the TDAE-initiated reactivity with the 4-chloroquinazoline scaffold. In addition to the description of two particular reactivities related to its basic and nucleophilic characters, this study showed the possibility to carry out aromatic nucleophilic reactions (SNAr). The synthesis of several 4-benzyl-2-trichloromethylquinazolines using this original methodology was related to a program of new antiplasmodial agents pharmacomodulation.

Conserved residues in protein are crucial for maintaining structure and function, either by direct involvement in chemistry or indirectly, by being essential for folding, stability and oligomerisation and are mostly clustered near active sites. The variability of sequences of the same protein from diverse organisms is a reflection of the selective pressures of evolution. Sequence conservation analysis with 3397 bacterial triosephosphate isomerase (TIM) sequences using Plasmodium falciparum (Pf) TIM as template, showed full conservation of ten residues, K12, T75, H95, E97, C126, E165, P166, G209, G210 and G228. The integrity of the enzyme active site, which lies near the dimer interface, makes TIM an obligatory dimer. Attempts to engineer active monomeric TIM have not been successful. The present study assesses the effects of mutations at fully conserved position 75 (Thr) and the highly conserved position 64 (Q: 3011, E: 383) near the dimer interface, using the recombinant Plasmodial enzyme. Residue 64, Gln in Pf, and T75 interact with the catalytic E97 and K12, respectively. Preliminary analysis of available crystal structures showed that Gln 64 takes part in a single intersubunit interaction and maintains the obligatory strained backbone angles of the catalytic K12 residue, while Thr 75 is involved in four intersusunit hydrogen bond interactions. This led to the hypothesis that mostly, Gln at position 64 is crucial for enzyme activity and Thr at position 75 for the integrity of the dimer. Biophysical and kinetic data are reported for four T75 (T75S/V/C/N) and two Q64 (Q64N/E) mutants. The major findings revealed that the mutations at position 64 have a significant effect on dimer integrity with a 1000 fold increase in the dimer dissociation constant compared to the wild type enzyme, while dimer stability was unimpaired for the T75 mutants. Concentration dependence of activity yielded an estimate of dimer dissociation constant (Kd) values (Q64N 73.7±9.2 nM and Q64E 44.6±8.4 nM). Enzyme activity values of the T75 mutants are comparable to the wild type, except for T75N which shows a 4-fold drop in activity. All four T75 mutants show a dramatic fall in activity between 35 °-45 °C. Crystal structure determination of the T75S/V/N mutant offers insights into the variation in local interactions with T75N showing the largest changes. These results were unanticipated emphasising the uncertainties involved in inferring functional and structural role for individual residues based only on analysis of interactions observed in crystal structures. Nanospray ionisation mass spectrometric studies has also been used to probe the oligomeric properties of the three mutant proteins Q64N, Q64E and T75S and the wild type enzyme in the gas phase. The gas phase distributions of dimeric and monomeric species have been examined under a wild range of collision energies (40 – 160 eV). The order in the gas phase, PfTIM wild type > T75S > Q64E ~ Q64N, together with the solution phase experiments described above establish the importance of Q64 and T75 in influencing stability and activity. Inhibition studies with a 27 residue synthetic dimer interface peptide and the Q64 mutants establish that the interaction between the protein and the peptide was facilitated in the case of monomeric species.

MSc (Chemistry) / Department of Chemistry / Malaria, an infectious disease affecting both human beings and other animals, is transmitted by parasitic protozoans belonging to the Plasmodium genus. Malaria is commonly treated with drugs such as quinine, chloroquine, and artesunate. However, the incidence of treatment failure due to drug-drug interactions and parasite resistance is increasing. Therefore, the rich medicinal potential of plants found in nature in Africa is increasingly being explored. The traditional use of Lippia javanica, Sclerocarya birrea, Melia azedarach and Capparis tomentosa for the treatment of malaria is well-known, but the phytochemistry of these four plants is not fully known. Parts of these plants were extracted and column chromatography was used to fractionate the extracts. The antioxidant activities of the fractions were determined using free radical scavenging and reducing power assays, while the cytotoxic, antiplasmodial and antitrypanosomal activities were determined using cell toxicity assay, parasite lactate dehydrogenase (pLDH) and trypanosome assay. The methanol stem bark extract of Melia azedarach (Fraction 2) had the highest phenolic content (59.39 mg GAE/g), while the methanol leaf extract of Melia azedarach had the highest flavonoid content of 188.65 mg QE/g. In the reducing power tests and DPPH free radical scavenging activity, the methanol stem bark extract of Melia azedarach had the lowest IC50 value of 0.1074 μg/mL and an IC0.5 value of 0.5296 μg/mL, respectively. Furthermore, the methanol stem bark extract of Melia azedarach at a concentration of 50 μg/mL showed significant cytotoxicity against HeLa cells (-1.22±0.07 %). The methanol stem bark extract of Melia azedarach at the tested concentration (250 μg/mL) decreased the viability of Plasmodium falciparum to 36.38±11.96 % with an IC50 value of 6.5 μg/mL. Concerning the antitrypanosomal activity, the methanol stem bark extract of Melia azedarach affected the viability of the trypanosomes at the tested concentration (250 μg/mL), giving a viability of 14.05 ± 0.59 %, with an IC50 value of 0.4 μg/mL. The presence of epicatechin (29) and catechin (31) in this extract was confirmed using several spectroscopic techniques (IR, NMR, UPLC-MS and HRMS). / NRF

L'objectif de ce travail consiste en la synthèse et l'évaluation antiparasitaire in vitro de nouveaux dérivés du thiazole et apparentés. Plusieurs stratégies de synthèse visant à une pharmacomodulation en séries mono- et polycycliques ont été étudiées : en série 2-méthyl-5-nitrothiazole, la pharmacomodulation anti-Trichomonas de la position 4 par stratégie SRN1 n'a pas permis d'améliorer l'activité déjà démontrée en série 2-méthyl-5-nitroimidazole, mais a conduit à des dérivés à activité antiproliférative in vitro, spécifique de la lignée HepG2. En série 4-arylsulfonylméthyl-2-méthylthiazole, la pharmacomodulation de la position 5, par couplage de Suzuki-Miyaura d'une part, et par arylation directe et réaction de Knoevenagel intramoléculaire d'autre part, a conduit à des dérivés mono- et polycycliques dont certains ont démontré une activité antiplasmodiale in vitro encourageante. En série 5H-thiazolo[3,2-a]pyrimidin-5-one, la réaction de double couplage de Suzuki-Miyaura a révélé l'importance du groupement phényle en position 6 pour l'activité antiplasmodiale de ces dérivés. Enfin, l'évaluation biologique in vitro de thiéno[3,2-d]pyrimidin-4(3H)-ones a permis de caractériser le pharmacophore responsable de l'activité antiplasmodiale significative de cette série. Les résultats préliminaires encourageants d'une étude mécanistique antiplasmodiale présentent l'inhibition spécifique des kinases plasmodiales comme un mécanisme d'action potentiel de ces composés. / The objective of this work consists of the synthesis and the antiparasitic in vitro evaluation of new thiazole derivatives and related structures. Several synthetic strategies aiming at the pharmacomodulation on mono- and polycyclic series have been studied: in 2-methyl-5-nitrothiazole series, the anti-Trichomonas pharmacomodulation on position 4 by SRN1 strategy did not improve the activity previously demonstrated in 2-methyl-5-nitroimidazole series, but led to derivatives displaying a selective in vitro antiproliferative activity toward the HepG2 cell line. In 4-arylsulfonylmethyl-2-methylthiazole series, the pharmacomodulation on position 5, by Suzuki-Miyaura cross-coupling reaction on the one hand, and by direct arylation and intramolecular Knoevenagel reaction on the other hand, led to mono- and polycyclic derivatives among which some displayed an encouraging in vitro antiplasmodial activity. In 5H-thiazolo[3,2-a]pyrimidin-5-one series, a double Suzuki-Miyaura cross-coupling reaction revealed that the phenyl group on position 6 contributes to the antiplasmodial effect of this series. Finally, the in vitro biological evaluation of the thieno[3,2-d]pyrimidin-4(3H)-one scaffold let to characterize the pharmacophore responsible for the significant antiplasmodial activity. Some preliminary encouraging results regarding a mechanistic antiplasmodial study show the specific inhibition of plasmodial kinases, as a potential mechanism of action of some of these compounds.