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Nemaline myopathy is a subtype of congenital myopathy that is clinically characterized by muscle weakness and early hypotonia of variable severity. Pathologically, nemaline myopathy is characterized by the presence of nemaline rods that stain purple in modified Gӧmӧri trichrome dye in patient biopsies under a microscope. Affected individuals experience skeletal muscle weakness and feeding difficulties, but most individuals will also experience respiratory muscle weakness that is disproportional to the weakness in skeletal muscles. Currently, 6 different subtypes of nemaline myopathy have been identified, each caused by mutations in ACTA1, NEB, TPM2, TPM3, TNNT1, KBTBD13, CFL2, KLHL40, KLHL41, or LMOD3, which are genes that encode either thin filament proteins or Kelch-like proteins. Of these genes, mutations in NEB and ACTA1 account for the majority of nemaline myopathy cases. Due to the genetic heterogeneity of nemaline myopathy, it is imperative to discover therapeutic targets and treatments that can universally treat nemaline myopathy patients. Preliminary data from our lab has demonstrated that proteasome complexes are downregulated in nemaline myopathy patients. Further, proteasomal activators improved motor function in neb zebrafish models, demonstrating the potential for proteasome activators to be therapeutics for nemaline myopathy patients. To extend these studies, the effect of proteasome activators, betulinic acid and Rolipram, was evaluated on the motor function in neb zebrafish models. However, in our experimental trials with betulinic acid and Rolipram, no positive effect on motor function in neb zebrafish was observed. In order to confirm our findings for both betulinic acid and Rolipram, additional trials will need to be conducted. / 2019-10-31T00:00:00Z

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Diabetes and dyslipidemia prevalence has been increasing globally configured as an epidemic resultant mainly from overweight, physical inactivity and genetic susceptibility. There are reports of many natural products that have hypoglycemic and hypolipidemic activity. Among them we mention the terpenes, which are the largest group of secondary products of plants metabolism. The terpene studied in this work was betulinic acid (BA), a pentacyclic triterpene lupano type that presents a variety of biological and pharmacological activities. The objective of this study was to evaluate BA hypoglycemic and hypolipidemic effects in experimental protocols of dyslipidemia and diabetes induced pharmacologically as well as studying their toxic potential in vivo. The BA-treated groups received doses of 5 (BA5), 10 (BA10) and 20 (BA20) mg/kg. The evaluation of the BA hypoglycemic action was carried out through diabetes induced by alloxan protocol and oral glucose tolerance test (OGTT). To check their activity on lipid metabolism, it was carried out dyslipidemia protocol induced by Triton WR 1339 intraperitoneal injection. In addition, it was performed the protocol modified diet-induced hypercholesterolemia. Toxicity was assessed by the study repeated doses for 28 days treating daily with BA doses via gavage. After the protocol of alloxan-induced diabetes, there was a blood glucose reduction in groups BA10 and BA20. Triglycerides and total cholesterol decreased significantly at all doses studied. BA10 treatment, also reduced the blood glucose peak caused by glucose overload (2g/Kg) in the OGTT. After 24 hours of dyslipidemia induced by triton, there was a significant triglycerides reduction in groups treated with BA at doses of 10 and 20mg/Kg. After 48h, triglycerides levels remained reduced in group treated with BA10. In hypercholesterolemia induced by diet modified protocol BA at doses of 10 and 20mg/kg, promoted a significant decrease in total cholesterol levels. There were no significant changes in the parameters evaluated after repeated dose oral toxicity protocol. Results demonstrate the therapeutic potential and safety of betulinic acid in the treatment of dyslipidemia and diabetes, although others pre-clinical and clinical studies are necessary for its use by population. / A prevalÃncia da diabetes e das dislipidemias vem crescendo mundialmente configurando-se como uma epidemia resultante,principalmente, do excesso de peso, da inatividade fÃsica e da suscetibilidade genÃtica. Existem relatos de muitos produtos de origem natural que possuem atividade hipoglicÃmica e hipolipidÃmica. Dentre eles, podemos citar os terpenos, que constituem o maior grupo de produtos do metabolismo secundÃrio de plantas. O terpeno estudado no presente trabalho à o Ãcido betulÃnico (AB), um triterpeno pentacÃclico do tipo lupano que apresenta uma variedade de atividades biolÃgicas e farmacolÃgicas. Assim, o objetivo deste estudo foi avaliar o efeito hipolipidÃmico e hipoglicÃmico do AB em protocolos experimentais de dislipidemias e diabetes induzidas farmacologicamente bem como estudar seu potencial tÃxico in vivo. O grupos tratados com AB receberam as doses de 5(AB5), 10(AB10) e 20(AB20)mg/Kg. A avaliaÃÃo da aÃÃo do hipoglicÃmica do AB foi atravÃs do protocolo de diabetes induzida por aloxano e o teste oral de tolerÃncia a glicose (TOTG). Para verificar sua atividade sobre o metabolismo lipÃdico, foi realizado o protocolo de induÃÃo da dislipidemia atravÃs da injeÃÃo intraperitoneal de triton WR 1339. AlÃm disso, foi realizado o protocolo de hipercolesterolemia induzida por dieta modificada. A toxicidade foi avaliada pela realizaÃÃo do estudo toxicolÃgico de doses repetidas durante 28 dias mediante administraÃÃo Ãnica diÃria por via oral de AB. Depois do protocolo de diabetes induzida por aloxano, observou-se uma reduÃÃo da glicemia nos animais dos grupos AB10 e AB20. Os triglicerÃdeos e o colesterol total reduziram significativamente em todas as doses estudas. O tratamento com AB10, reduziu ainda o pico glicÃmico causado pela sobrecarga de glicose (2g/Kg) no TOTG. ApÃs 24h da induÃÃo com triton, verificou-se a reduÃÃo significativa dos triglicerÃdeos nos grupos tratados com AB nas doses de 10 e 20mg/Kg. Depois de 48h, os animais do grupo AB10 manteve tal reduÃÃo. No protocolo de hipercolesterolemia induzida por dieta modificada o AB nas doses de 10 e 20mg/Kg, promoveu uma diminuiÃÃo significativa do colesterol total plasmÃtico. NÃo foram encontradas alteraÃÃes significativa nos parÃmetros avaliados apÃs protocolo de toxicidade oral em doses repetidas. Os resultados obtidos demonstram o potencial terapÃutico e a seguranÃa do Ãcido betulÃnico no tratamento das dislipidemias e diabetes, apesar de serem necessÃrios novos estudos prÃ-clÃnicos e clÃnicos para sua utilizaÃÃo no mercado.

Tolfenamic acid (TA), betulinic acid (BA) and acetylsalicylic acid (aspirin) are anticancer drugs, and Sp1, Sp3, Sp4 transcription factors and growth factor 2 (EGFR2, HER2 / ErbB2) are important molecular markers in cancer cells. In this study the molecular mechanisms by which these anticancer drugs target downregulation of Sp1, Sp3, Sp4 and ErbB2 are investigated in breast cancer cells. TA inhibits growth of ErbB2-overexpressing BT474 and SKBR3 breast cancer cells by inhibiting ErbB2 expression. In cells treated with TA, ErbB2 mRNA expression and promoter activity were decreased, and this was due to decreased mRNA stability in BT474 cells. In both cell lines, TA also decreased expression of the YY1 and AP-2 transcription factors that are required for basal ErbB2 expression. These effects were accompanied by decreased ErbB2-dependent kinase activities, induction of p27, and decreased expression of cyclin D1. In addition, TA also inhibited tumor growth in BT474 cells orthotopic mouse model. However, Sp proteins were not the major target of TA in these breast cancer cells and this contrasts to results in pancreatic cancer cells where TA decreased expression of Sp proteins. BA inhibits growth of ErbB2 –overexpressing BT474 and MDA-MB-453 and induced apoptosis in these cells. BA induced proteasome-independent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and survivin, an Sp-regulated gene, and BA also decreased expression of ErbB2, ErbB2-regulated kinases and also YY1, a transcription factor that regulates ErbB2 expression in these cells. Knockdown of Sp1, Sp3, Sp4 and their combination by RNA interference was accompanied by decreased expression of ErbB2, YY1 and luciferase activity in cells transfected with a construct containing the GC-rich YY1 promoter linked to a luciferase reporter gene. BA-dependent repression of Sp1, Sp3, Sp4 and Sp regulated genes was due in part to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a) which constitutively inhibits ZBTB10 expression. The effects of BA on the miR-27a:zBTB10-Sp transcription factor axis were inhibited in cells cotreated with the cannabinoid 1 (CB1) and CB2 receptor antagonists AM251 and AM630 respectively. However, in vitro binding studies with ≤ 10 mM BA and a radiolabeled cannabinoid did not indicate direct competitive binding of BA to the CB1 and CB2 receptors, suggesting a possible role for other CB-like G protein-coupled receptors. Aspirin inhibits growth, induces apoptosis and decreases cell migration in BT474 and MDA-MB-453 breast cancer cells that overexpress the ErbB2 oncogene. Aspirin also downregulated ErbB2 expression in these cells and this was accompanied by inhibition of downstream kinases including phospho-Akt (p-Akt) and phospho-mitogen-activated protein kinase (p-MAPK). Aspirin also decreased expression of survivin, vascular endothelial growth factor (VEGF) and YY1 which regulates ErbB2 expression in these cell lines. Aspirin also downregulates Sp1, Sp3 and Sp4, and further investigation of the underlying mechanism of action showed that aspirin-induced downregulation of Sp transcription factors and Sp-regulated genes could be inhibited in part by proteasome inhibitior lactacystin and phosphatase inhibitors including sodium orthovanadate (SOV). These results were consistant with the induction of several phosphatases by aspirin in BT474 and MDA-MB-453 cells and these include mitogen-activated protein kinase phosphatase-5 (MKP-5) and MKP-1. Aspirin-induced downregulation of Sp1, Sp3 and Sp4 are reversed in cells transfected with an oligonucleotide (siMKP5) that knocks down MKP5 by RNA interference whereas siMKP-1 did not block Sp protein downregulation demonstrating for the first time a linkage between a drug-induced phosphatase (MKP-5) and Sp downregulation. These results suggest that TA, BA and aspirin represent novel and promising new anticancer drugs for cancer treatment by targeting Sp proteins and ErbB2 oncogene.

Master of Science (Nanoscience) / Neuroblastomas (NBs) are the most common solid extra-cranial tumours diagnosed in childhood and characterized by a high risk of tumour relapse. Like in other tumour types, there are major concerns about the specificity and safety of available drugs used for the treatment of NBs, especially because of potential damage to the developing brain. Many plant-derived bioactive compounds have proved effective for cancer treatment but are not delivered to tumour sites in sufficient amounts due to compromised tumour vasculature characterized by leaky capillary walls. Betulinic acid (BetA) is one such naturally-occurring anti-tumour compound with minimum to no cytotoxic effects in healthy cells and rodents. BetA is however insoluble in water and most aqueous solutions, thereby limiting its therapeutic potential as a pharmaceutical product. Liposomes are self-assembling closed colloidal structures composed of one or more concentric lipid bilayers surrounding a central aqueous core. The unique ability of liposomes to entrap hydrophilic molecules into the core and hydrophobic molecules into the bilayers renders them attractive for drug delivery systems. Cyclodextrins (CDs) are non-reducing cyclic oligosaccharides which proximate a truncated core, with features of a hydrophophilic outer surface and hydrophobic inner cavity for forming host-guest inclusion complexes with poorly water soluble molecules. CDs and liposomes have recently gained interest as novel drug delivery vehicles by allowing lipophilic/non-polar molecules into the aqueous core of liposomes, hence improving the therapeutic load, bioavailability and efficacy of many poorly water-soluble drugs. The aim of the study was to develop nano-drug delivery systems for BetA in order to treat human neuroblastoma (NB) cancer cell lines. This was achieved through the preparation of BetA liposomes (BetAL) and improving the percent entrapment efficiency (% EE) of BetA in liposomes through double entrapment of BetA and gamma cyclodextrin BetA inclusion complex (γ-CD-BetA) into liposomes (γ-CD-BetAL). We hypothesized that the γ-CD-BetAL would produce an increased % EE compared to BetAL, hence higher cytotoxic effects. Empty liposomes (EL), BetAL and γ-CD-BetAL were synthesized using the thin film hydration method followed by manual extrusion. Spectroscopic and electron microscopic characterization of these liposome formulations showed size distributions of 1-4 μm (before extrusion) and less than 200 nm (after extrusion). As the liposome size decreased, the zeta-potential (measurement of liposome stability) decreased contributing to a less stable liposomal formulation. Low starting BetA concentrations were found to be more effective in entrapping higher amounts of BetA in liposomes while the incorporation of γ-CD-BetA into liposomes enhanced the % EE when compared to BetAL, although this was not statistically significant. Cell viability studies using the WST-1 assay showed a time-and concentration-dependent decrease in SK-N-BE(2) and Kelly NB cell lines exposed to free BetA, BetAL and γ-CD-BetAL at concentrations of 5-20 ug/ml for 24, 48 and 72 hours treatment durations. The observed cytotoxicity of liposomes was dependant on the % EE of BetA. The γ-CD-BetAL was more effective in reducing cell viability in SK-N-BE(2) cells than BetAL whereas BetAL was more effective in KELLY cells at 48-72 hours. Exposure of all cells to EL showed no toxicity while free BetA was more effective overall than the respective liposomal formulations. The estimated IC₅₀ values following exposure to free BetA and BetAL were similar and both showed remarkable statistically significant decrease in NB cell viability, thus providing a basis for new hope in the effective treatment of NBs.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-30T21:16:53Z No. of bitstreams: 1 Matheus Santos de Sá Avaliacao da atividade antimalarica de substancias obtidas de especies vegetais....pdf: 9210254 bytes, checksum: 6b1ee3754729e2a51460a41ce1709423 (MD5) / Made available in DSpace on 2012-07-30T21:16:53Z (GMT). No. of bitstreams: 1 Matheus Santos de Sá Avaliacao da atividade antimalarica de substancias obtidas de especies vegetais....pdf: 9210254 bytes, checksum: 6b1ee3754729e2a51460a41ce1709423 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A malária é uma das mais importantes infecções parasitárias de seres humanos devido à alta morbidade e mortalidade atribuídas a esta doença, que constitui uma ameaça para mais de dois bilhões de pessoas vivendo nas áreas de alta incidência. O Plasmodium falciparum, um dos agentes causadores da malária, apresenta alta capacidade de adaptação por mutação e pode ser resistente a vários tipos de drogas antimaláricas já disponíveis, como a cloroquina, o que torna importante a busca de novos antimaláricos. A região do semi-árido brasileiro abrange cerca de 11,5% do território nacional, e possui o bioma menos estudado em relação à flora e fauna, e um dos que tem sofrido maior degradação pelo uso desordenado e predatório nos últimos 400 anos. Tendo em vista o potencial farmacológico dos produtos naturais, o objetivo desse trabalho foi avaliar a atividade antimalárica de substâncias puras extraídas de espécies vegetais nativas ou endêmicas do semi-árido brasileiro e derivados sintéticos. A partir de uma biblioteca de 160 substâncias triadas para atividade antimalárica, foram selecionadas duas classes de compostos para avaliações in vitro e in vivo: o ácido betulínico e derivados, bem como o lapachol e derivados. Foi selecionada ainda uma terceira classe de moléculas, as fisalinas, utilizando o método do Similarity Ensemble Approach (SEA), que previu a ação antimalárica dessas substâncias. Dentre os derivados do ácido betulínico testados, o acetato do ácido betulínico apresentou a maior potência farmacológica in vitro quando comparado com os outros derivados, e foi ativo in vivo. A atividade antimalárica das fisalinas foi confirmada em ensaios in vitro. Ao serem analisadas in vivo, as fisalinas F e D apresentaram resultados opostos (exacerbação e proteção contra a infecção, respectivamente), possivelmente devido à atividade imunossupressora da fisalina F e ausente na fisalina D. A análise do lapachol e seus derivados iniciou-se através de estudos in silico por Quantitative Structure-Activity Relationship (QSAR), que indicaram ser o isolacet o derivado com maior atividade, o que foi confirmado por ensaios in vitro. A atividade antimalárica do isolacet foi confirmada in vivo, sendo ainda realizados estudos de Docking desta molécula com a falcipaína 2 de P. falciparum, que indicaram ser esta cisteíno-protease um possível alvo do isolacet. Nossos resultados indicam o potencial antimalárico de compostos isolados a partir de plantas do semi-árido e demonstram a importância da associação de várias abordagens para entendimento dos mecanismos de ação de moléculas com atividade farmacológica. / Malaria is one of the most important parasitic infections of humans due to the high morbidity and mortality attributed to this disease, which threatens to over two billion people living in areas with high incidence. Plasmodium falciparum, a causative agent of malaria, has a high capacity to adapt by mutation and may be resistant to various antimalarial drugs already available, such as chloroquine, which makes it important to search for new antimalarials. The Brazilian semi-arid region cover about 11.5% of the country, and the biome has been less studied in relation to flora and fauna, and one who has suffered further degradation and predation by the inordinate use in the last 400 years. Given the pharmacological potential of natural products, the aim of this study was to evaluate the antimalarial activity of pure compounds extracted from native or endemic plant species of arid and semi-synthetic derivatives. From a library of 160 substances screened for antimalarial activity, we selected two classes of compounds for evaluation in vitro and in vivo: The betulinic acid and derivatives, as well as lapachol and derivatives. It was also selected a third class of molecules, physalins using the method of Similarity Ensemble Approach (SEA), who predicted the antimalarial action of these substances. Among the tested derivatives of betulinic acid, betulinic acid acetate showed the highest pharmacological potency in vitro when compared with other derivatives, and was active in vivo. The antimalarial activity of physalins was confirmed in vitro assays. When analyzed in vivo the physalins F and D had the opposite results (exacerbation and protection against infection, respectively), possibly due to the immunosuppressive activity of physalin F and absent in physalin D. The analysis of lapachol and its derivatives was initiated through studies in silico by Quantitative Structure-Activity Relationship (QSAR), which indicated that the isolacet the derivative with greater activity, which was confirmed by in vitro assays. The antimalarial activity of isolacet was confirmed in vivo, and further studies of this molecule by Docking with falcipain 2 P. falciparum, which indicated that this cysteine protease is a possible isolacet target. Our results indicate the potential antimalarial compounds isolated from plants of the semi-arid and demonstrate the importance of the combination of various approaches to understanding the mechanisms of action of molecules with pharmacological activity.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-03-17T18:30:52Z No. of bitstreams: 1 Nóbrega, Andrea Bezerra da [Dissertação, 2012].pdf: 4003660 bytes, checksum: 8ad06bfbfef99e402671fe62870055a8 (MD5) / Made available in DSpace on 2017-03-17T18:30:52Z (GMT). No. of bitstreams: 1 Nóbrega, Andrea Bezerra da [Dissertação, 2012].pdf: 4003660 bytes, checksum: 8ad06bfbfef99e402671fe62870055a8 (MD5) / O objetivo deste trabalho foi padronizar os extratos de Eugenia florida DC. e estudar suas atividades farmacológicas visando o desenvolvimento de um quimioterápico de origem vegetal. Para tanto, foram realizados estudos com diferentes métodos de extração, estudo de variação metabólica da planta, desenvolvimento e validação de metodologias analíticas, ensaios citotóxicos e ensaios em células tumorais. Os dados obtidos no trabalho apontaram a percolação no tempo de 2 horas como o método que apresentou o melhor percentual de extração de ácido betulínico a partir das folhas de E. florida. Apesar de saber que a granulometria influencia diretamente na eficiência da extração, não foram observadas grandes diferenças percentuais nos teores de ácido betulínico para os diferentes tamanhos de partículas utilizados na extração por ultrassom, porém constatou-se que a homogeneidade dos tamanhos de partículas aumenta a eficiência da extração. No estudo de variação metabólica observaramse variações durante o ano que devem ser consideradas na qualidade da matéria-prima vegetal. Os extratos de E. florida apresentaram baixa citotoxidez em células VERO e apresentaram índice de seletividade alto o que representa uma maior afinidade pelas células tumorais do que pelas células sadias (VERO). Nos ensaios com células tumorais os extratos apresentaram atividade mais potente contra a linhagem de ovário (OVOCAR-3), muito mais que os padrões de ácido betulínico (comercial e isolado), o que confirma a presença de outra(s) substância(s) com atividade antitumoral presente(s) no extrato o que corrobora para a pesquisa e o desenvolvimento de um fitoterápico ao invés de um fitofármaco para ser utilizado no tratamento do câncer de ovário / The objective of this study was the standardize of Eugenia florida DC. leaves extracts and study their pharmacological activities aiming the development of a chemoterapic. To achieve this goal, studies were performed using different particle sizes, metabolic profile, as well as development and validation of analytical methodologies, cytotoxic assays and assays on tumor cells. The data obtained on this study showed that the percolation extraction method in the time of 2 hours showed the better rate of betulinic acid extraction from E. florida leaves. Despite the knowledge that the particle size directly affects the efficiency of ultrasound extraction, major differences were not observed in the percentage of betulinic acid for the different particle sizes used, however the uniformity of the particle size increases the efficiency of extraction. In the metabolic profile study, variations were observed during the year which should be considered in the quality of the vegetable raw material. E. florida extracts showed lower citotoxicity in VERO cells, and showed high selectivity index which represents a greater affinity for tumor cells than for healthy cells. In tumoral cells assays, the extracts showed stronger activity against the ovarian line (OVOCAR-3), much more than the standards of betulinic acid (commercial and isolated), which confirms the influence of (an) other substance (s) presented in the extract with antitumoral activity in the extract which defines the development of a herbal medicine instead of a herbal drug to be used for the treatment of ovarian cancer

Esta tese alia dois enfoques principais dentro da Química Farmacêutica. Por um lado, busca explorar uma nova fonte de insumos naturais, cascas de Malus  domestica, com vistas a obtenção dos triterpenos ácidos ursólico (AU) e betulínico (AB); e por outro lado, estuda a relação dos derivados triterpênicos semissintéticos obtidos com a proteína-alvo (PfATP6) destacada atualmente na literatura da terapia da malária com vistas ao planejamento de novos antimaláricos. O primeiro Capítulo inclui o desenvolvimento de um método eficiente, fácil e extremamente rápido onde são combinadas as técnicas de extração por ultrassom (UAE) e análise por Cromatografia Líquida de Alta Eficiência Acoplada a Detector de Espectroscopia de Massas (LC-MS), para identificação e doseamento dos ácidos ursólico (AU) e betulínico (BA) em extratos de cascas frescas de maçã de cinco clones das cultivares Gala e Fuji (“Baigent”, “Fuji Mishima”, “Fuji Suprema”, “Fuji Select” and “Maxi Gala”) oriundas da Região Sul do Brasil. Os parâmetros cromatográficos do método analítico incluem: ionização por eletro spray em modo positivo (ESI+), fluxo de 1,0 mL/min, em modo de eluição isocrático, consistindo de 80% acetonitrila e 20% acetato de amônio 10 mM em pH 6,0 e temperatura ambiente. O método desenvolvido foi validado e mostrou ser seletivo, sensível (LOD e LOQ de 0,087 e 0,266 μg/mL para BA, e 0,398 e 2,117 μg/ mL para UA), coeficiente de regressão linear (r >0.99), preciso, exato e robusto para os analitos de interesse. A otimização do método combinado de UAE com LC-MS permitiu concluir os procedimentos de extração e análise em tempo inferior a 4 h, uma vez que o método não requer a secagem da amostra, etapa que demanda longos tempos de processamento. Este método foi aplicado e forneceu a primeira caracterização fitoquímica dos cinco clones de maçã estudados. Os resultados demonstraram que o método combinado de UAE-LC-MS é adequado às práticas do controle de qualidade. O segundo Capítulo apresenta o estudo da interação dos ligantes semissintéticos derivados dos AU e AB, sintetizados pelo nosso grupo de pesquisa, com a proteína Ca2+-ATPase do Plasmodium falciparum (PfATP6), através do emprego da técnica de Docking Molecular. A PfATP6 é descrita como um importante alvo para novos antimaláricos como Artemisinina (ART), cujo mecanismo de ação inclui, dentre outros, a modulação da homeostasia do cálcio intracelular. Investigações conduziram à hipótese do extravasamento do cálcio, do interior do retículo sarco-endoplasmático como mecanismo plausível para ação dos derivados triterpênicos do AU e AB. Os escores de energia determinados no Docking, de cada um dos nove ligantes (derivados triterpênicos) e quatro compostos de controle (ácidos ursólico e betulínico, artemisinina (ART) e tapsigargina (TPG) foram determinados (análises realizadas em triplicata) e correlacionados com os valores de IC50, para a atividade antimalárica. Os resultados mostraram excelente correlação entre os escores de energia (com a PfATP6) e os valores de IC50, superior a 80% (r > 0,83360). O estudo fornece fortes evidências de que a PfATP6 pode constituir um alvo para os derivados pentacíclicos do estudo, bem como permitiu identificar o perfil conformacional dos ligantes e os principais resíduos do sítio de ligação (SL) da PfATP6 envolvidos nas interações; bem como, contribuiu para a melhor compreensão das propriedades envolvidas na interação do ligante com o receptor e mecanismo de ação. O terceiro Capítulo faz uso de Métodos Clássicos e Quânticos para descrever as mudanças conformacionais da proteína PfATP6. As simulações de Dinâmica Molecular do receptor na forma isolada e complexada com os ligantes foram realizadas durante 10 ns. As conformações finais obtidas para os receptores foram avaliadas em termos RMSD, efeito da presença dos ligantes no sítio de ligação e tipos de interações estabelecidas entre o ligante e o receptor. Os resultados mostraram que as proteínas PfATP6 e SERCA tendem a manter sua conformação nativa e que o modelo utilizado é adequado aos propósitos do estudo. O monitoramento dos resíduos da região citoplasmática das proteínas permitiu evidenciar o efeito alostérico da presença dos ligantes AU e ART no SL, sobre os domínios A e N, da PfATP6. Esse efeito reproduz as conformações E1 e E2, bem estabelecidas para PfATP6, na presença e ausência de Ca2+. As análises de Dinâmica Molecular corroboram os achados do Capítulo II ao evidenciarem o estabelecimento de interações de hidrogênio com os resíduos importantes do SL da PfATP6. Esses resultados fundamentam os indícios de que as bombas de Ca2+-ATPase (SERCA), possam ser de fato, um alvo para os derivados triterpênicos dos AU e AB. / This thesis combines two main focuses within Pharmaceutical Chemistry. On the one hand, it seeks to explore a new source of natural inputs, bark of Malus domestica, in order to obtain ursolic (UA) and betulinic acid (BA) triterpenes. On the other hand, it studies the relation of the semi-synthetic triterpenic derivatives obtained with the target proteins (PfATP6), currently highlighted in the literature on malaria therapy with a view to planning new antimalarial drugs. The first chapter includes the development of an efficient, easy and extremely fast method where ultrasonic extraction techniques (UAE) and high-performance liquid chromatography coupled to mass spectroscopy (LC-MS) are combined for identification and assay of ursolic acid (UA) and betulinic acid (BA) in fresh apple peel extracts from five clones of the Gala and Fuji cultivars (Baigent, Fuji Mishima, Fuji Suprema, Fuji Select and Maxi Gala ") in the Southern Region of Brazil. Chromatographic parameters of the analytical method include: electrospray ionization (ESI +), flow rate of 1.0 mL/min in isocratic elution mode, consisting of 80% acetonitrile and 20% 10 mM ammonium acetate at pH 6.0 and room temperature. The method was validated and proved to be selective, sensitive (LOD and LOQ of 0.087 and 0.266 μg/mL for BA, and 0.398 and 2.117 μg/mL for UA), linear regression coefficient (r> 0.99), accurate, robust for analytes of interest. The optimization of the combined method of UAE with LC-MS allowed to complete the procedures of extraction and analysis in less than 4 h, since the method does not require drying the sample, a stage that demands long processing times. This method was applied and provided the first phytochemical characterization of the five apple clones studied. The results demonstrated that the combined UAE-LC-MS method is suitable for quality control practices. The second chapter presents the study of the interaction of the semi-synthetic ligands derived from the UA and BA, synthesized by our research group, with the Plasmodium falciparum Ca2+-ATPase protein (PfATP6), using the Molecular Docking technique. PfATP6 is described as an important target for new antimalarials such as Artemisinin (ART), whose action mechanism includes, among others, the modulation of intracellular calcium homeostasis. Investigations led to the hypothesis of extravasation of calcium from the interior of the sarco-endoplasmic reticulum as a plausible mechanism for the action of the triterpenic derivatives of UA and BA. The Docking energy scores (binding energy) of each of the nine ligands (triterpenic derivatives) and four control compounds (ursolic and betulinic acids, artemisinin (ART) and tapsigargine (TPG)) with PfATP6, were calculated (analyses performed in triplicate) and correlated with its antimalarial IC50 value. The results showed an excellent correlation between energy scores (with PfATP6) and IC50 values, higher than 80% (r > 0.83360). The study supplies strong evidence that PfATP6 may be a target for the pentacyclic derivatives of the study, and also allowed identifying the conformational profile of the ligands and the main residues of the PfATP6 binding site (BS) involved in the interactions. It further contributed to a better understanding of the properties involved in the interaction of the ligand with the receptor and its action mechanism. The third chapter uses Classical and Quantum Methods to describe the conformational changes of the PfATP6 protein. The Molecular Dynamics simulations of the receptor in the isolated and complexed form with the ligands were performed for 10 ns. The final conformations obtained for the receptors were evaluated in RMSD terms, effect of the presence of ligands at the binding site and types of interactions established between the ligand and the receptor. The results showed that the PfATP6 and SERCA proteins tend to maintain their native conformations and that the model used is adequate for the purposes of the study. The monitoring of the residues of the cytoplasmic region of the proteins allowed evidencing the allosteric effect of the presence of UA and ART ligands in BS, on the A- and N-domains of PfATP6. This effect reproduces the well-established E1 and E2 conformations for PfATP6, in the presence and absence of Ca2+, respectively. Molecular Dynamics analyses corroborate the findings of Chapter II, by showing the possibility of establishing hydrogen interactions with the important residues of PfATP6 BS. These results support the evidence that Ca2+-ATPase (SERCA) pumps may be a target for the triterpenic derivatives of UA and BA.

Submitted by Natalie Mendes (nataliermendes@gmail.com) on 2015-07-28T00:17:52Z No. of bitstreams: 1 Disserta??o - C?ssio Santana.pdf: 10345998 bytes, checksum: 41cc3a308f7a72f1c4e10cc1ee2fe345 (MD5) / Made available in DSpace on 2015-07-28T00:17:52Z (GMT). No. of bitstreams: 1 Disserta??o - C?ssio Santana.pdf: 10345998 bytes, checksum: 41cc3a308f7a72f1c4e10cc1ee2fe345 (MD5) Previous issue date: 2014-02-05 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / Chagas disease is a zoonosis caused by the hemoflagellate protozoan Trypanosoma cruzi, which affects millions of people in Latin America. Disease treatment is based on the use of two drugs, benznidazole and nifurtimox, which present low cure rates in the chronic phase of the disease as well as generating a number of adverse side effects. In this context, the development of new therapies for a better treatment of Chagas disease is necessary. In this study we investigated the anti-T.cruzi potential of physalins and betulinic acid, plant-based isolated steroids, in addition to synthetic derivatives from the latter, in in vitro assays. Our results demonstrate a high trypanocidal activity of physalins B and F and of the derivative BA5 against trypomastigote forms and on the processes of invasion and development of these on peritoneal macrophages. Electron microscopy revealed that physalin B or BA5 derivative treatment resulted in ultrastructural changes in the plasma membrane, Golgi apparatus, kinetoplast and endoplasmic reticulum of trypomastigotes forms. Additionally, these compounds contributed to the formation of atypical vacuoles in the appearance of myelin figures, which were labeled with MDC to confirm their identity as autophagic vacuoles. Flow cytometry analysis revealed that parasite death occurred mainly by necrosis. When combined with benznidazole, physalin B, physalin F or BA5 derivative, resulted in a higher anti-T. cruzi activity against amastigotes when compared to the compounds tested alone. These results indicate that steroids, such as physalins B and F and the derivative BA5, are potential candidates for an alternative treatment of Chagas disease. / A doen?a de Chagas ? uma zoonose causada pelo protozo?rio hemoflagelado Trypanosoma cruzi, que afeta milh?es de pessoas na Am?rica Latina. O tratamento dessa enfermidade se baseia na utiliza??o de dois f?rmacos, o benzonidazol e o nifurtimox, que possuem baixa taxa de cura na fase cr?nica da doen?a, al?m de gerarem uma s?rie de efeitos colaterais. Nesse contexto, o surgimento de novos medicamentos para uma quimioterapia mais adequada da doen?a de Chagas torna-se necess?rio. Neste estudo investigamos o potencial anti-T. cruzi de esteroides isolados de plantas, fisalinas e ?cido betul?nico, assim como de derivados sint?ticos deste ?ltimo, em ensaios in vitro. Nossos resultados demonstram uma elevada atividade tripanocida das fisalinas B e F e do derivado BA5 contra formas tripomastigotas e sobre os processos de invas?o e desenvolvimento destas em macr?fagos peritoneais. Atrav?s de ensaios de microscopia eletr?nica foi poss?vel observar que o tratamento com a fisalina B ou com o derivado BA5 causa altera??es ultraestruturais na membrana plasm?tica, complexo de Golgi, cinetoplasto e ret?culo endoplasm?tico das formas tripomastigotas, al?m da forma??o de vac?olos at?picos e o aparecimento de figuras miel?nicas, que foram marcadas com MDC para confirmar a sua identidade como vac?olos autof?gicos. An?lises por citometria de fluxo revelaram que a morte parasit?ria ocorre principalmente por necrose. A combina??o da fisalina B, da fisalina F ou do derivado BA5 com o benzonidazol resultou em uma maior atividade anti-T. cruzi frente a formas amastigotas quando comparados aos compostos testados de forma isolada. Estes resultados indicam que esteroides, tais como as fisalinas B e F e o derivado BA5, s?o potencias candidatos para o tratamento alternativo da doen?a de Chagas.

Esta tese alia dois enfoques principais dentro da Química Farmacêutica. Por um lado, busca explorar uma nova fonte de insumos naturais, cascas de Malus  domestica, com vistas a obtenção dos triterpenos ácidos ursólico (AU) e betulínico (AB); e por outro lado, estuda a relação dos derivados triterpênicos semissintéticos obtidos com a proteína-alvo (PfATP6) destacada atualmente na literatura da terapia da malária com vistas ao planejamento de novos antimaláricos. O primeiro Capítulo inclui o desenvolvimento de um método eficiente, fácil e extremamente rápido onde são combinadas as técnicas de extração por ultrassom (UAE) e análise por Cromatografia Líquida de Alta Eficiência Acoplada a Detector de Espectroscopia de Massas (LC-MS), para identificação e doseamento dos ácidos ursólico (AU) e betulínico (BA) em extratos de cascas frescas de maçã de cinco clones das cultivares Gala e Fuji (“Baigent”, “Fuji Mishima”, “Fuji Suprema”, “Fuji Select” and “Maxi Gala”) oriundas da Região Sul do Brasil. Os parâmetros cromatográficos do método analítico incluem: ionização por eletro spray em modo positivo (ESI+), fluxo de 1,0 mL/min, em modo de eluição isocrático, consistindo de 80% acetonitrila e 20% acetato de amônio 10 mM em pH 6,0 e temperatura ambiente. O método desenvolvido foi validado e mostrou ser seletivo, sensível (LOD e LOQ de 0,087 e 0,266 μg/mL para BA, e 0,398 e 2,117 μg/ mL para UA), coeficiente de regressão linear (r >0.99), preciso, exato e robusto para os analitos de interesse. A otimização do método combinado de UAE com LC-MS permitiu concluir os procedimentos de extração e análise em tempo inferior a 4 h, uma vez que o método não requer a secagem da amostra, etapa que demanda longos tempos de processamento. Este método foi aplicado e forneceu a primeira caracterização fitoquímica dos cinco clones de maçã estudados. Os resultados demonstraram que o método combinado de UAE-LC-MS é adequado às práticas do controle de qualidade. O segundo Capítulo apresenta o estudo da interação dos ligantes semissintéticos derivados dos AU e AB, sintetizados pelo nosso grupo de pesquisa, com a proteína Ca2+-ATPase do Plasmodium falciparum (PfATP6), através do emprego da técnica de Docking Molecular. A PfATP6 é descrita como um importante alvo para novos antimaláricos como Artemisinina (ART), cujo mecanismo de ação inclui, dentre outros, a modulação da homeostasia do cálcio intracelular. Investigações conduziram à hipótese do extravasamento do cálcio, do interior do retículo sarco-endoplasmático como mecanismo plausível para ação dos derivados triterpênicos do AU e AB. Os escores de energia determinados no Docking, de cada um dos nove ligantes (derivados triterpênicos) e quatro compostos de controle (ácidos ursólico e betulínico, artemisinina (ART) e tapsigargina (TPG) foram determinados (análises realizadas em triplicata) e correlacionados com os valores de IC50, para a atividade antimalárica. Os resultados mostraram excelente correlação entre os escores de energia (com a PfATP6) e os valores de IC50, superior a 80% (r > 0,83360). O estudo fornece fortes evidências de que a PfATP6 pode constituir um alvo para os derivados pentacíclicos do estudo, bem como permitiu identificar o perfil conformacional dos ligantes e os principais resíduos do sítio de ligação (SL) da PfATP6 envolvidos nas interações; bem como, contribuiu para a melhor compreensão das propriedades envolvidas na interação do ligante com o receptor e mecanismo de ação. O terceiro Capítulo faz uso de Métodos Clássicos e Quânticos para descrever as mudanças conformacionais da proteína PfATP6. As simulações de Dinâmica Molecular do receptor na forma isolada e complexada com os ligantes foram realizadas durante 10 ns. As conformações finais obtidas para os receptores foram avaliadas em termos RMSD, efeito da presença dos ligantes no sítio de ligação e tipos de interações estabelecidas entre o ligante e o receptor. Os resultados mostraram que as proteínas PfATP6 e SERCA tendem a manter sua conformação nativa e que o modelo utilizado é adequado aos propósitos do estudo. O monitoramento dos resíduos da região citoplasmática das proteínas permitiu evidenciar o efeito alostérico da presença dos ligantes AU e ART no SL, sobre os domínios A e N, da PfATP6. Esse efeito reproduz as conformações E1 e E2, bem estabelecidas para PfATP6, na presença e ausência de Ca2+. As análises de Dinâmica Molecular corroboram os achados do Capítulo II ao evidenciarem o estabelecimento de interações de hidrogênio com os resíduos importantes do SL da PfATP6. Esses resultados fundamentam os indícios de que as bombas de Ca2+-ATPase (SERCA), possam ser de fato, um alvo para os derivados triterpênicos dos AU e AB. / This thesis combines two main focuses within Pharmaceutical Chemistry. On the one hand, it seeks to explore a new source of natural inputs, bark of Malus domestica, in order to obtain ursolic (UA) and betulinic acid (BA) triterpenes. On the other hand, it studies the relation of the semi-synthetic triterpenic derivatives obtained with the target proteins (PfATP6), currently highlighted in the literature on malaria therapy with a view to planning new antimalarial drugs. The first chapter includes the development of an efficient, easy and extremely fast method where ultrasonic extraction techniques (UAE) and high-performance liquid chromatography coupled to mass spectroscopy (LC-MS) are combined for identification and assay of ursolic acid (UA) and betulinic acid (BA) in fresh apple peel extracts from five clones of the Gala and Fuji cultivars (Baigent, Fuji Mishima, Fuji Suprema, Fuji Select and Maxi Gala ") in the Southern Region of Brazil. Chromatographic parameters of the analytical method include: electrospray ionization (ESI +), flow rate of 1.0 mL/min in isocratic elution mode, consisting of 80% acetonitrile and 20% 10 mM ammonium acetate at pH 6.0 and room temperature. The method was validated and proved to be selective, sensitive (LOD and LOQ of 0.087 and 0.266 μg/mL for BA, and 0.398 and 2.117 μg/mL for UA), linear regression coefficient (r> 0.99), accurate, robust for analytes of interest. The optimization of the combined method of UAE with LC-MS allowed to complete the procedures of extraction and analysis in less than 4 h, since the method does not require drying the sample, a stage that demands long processing times. This method was applied and provided the first phytochemical characterization of the five apple clones studied. The results demonstrated that the combined UAE-LC-MS method is suitable for quality control practices. The second chapter presents the study of the interaction of the semi-synthetic ligands derived from the UA and BA, synthesized by our research group, with the Plasmodium falciparum Ca2+-ATPase protein (PfATP6), using the Molecular Docking technique. PfATP6 is described as an important target for new antimalarials such as Artemisinin (ART), whose action mechanism includes, among others, the modulation of intracellular calcium homeostasis. Investigations led to the hypothesis of extravasation of calcium from the interior of the sarco-endoplasmic reticulum as a plausible mechanism for the action of the triterpenic derivatives of UA and BA. The Docking energy scores (binding energy) of each of the nine ligands (triterpenic derivatives) and four control compounds (ursolic and betulinic acids, artemisinin (ART) and tapsigargine (TPG)) with PfATP6, were calculated (analyses performed in triplicate) and correlated with its antimalarial IC50 value. The results showed an excellent correlation between energy scores (with PfATP6) and IC50 values, higher than 80% (r > 0.83360). The study supplies strong evidence that PfATP6 may be a target for the pentacyclic derivatives of the study, and also allowed identifying the conformational profile of the ligands and the main residues of the PfATP6 binding site (BS) involved in the interactions. It further contributed to a better understanding of the properties involved in the interaction of the ligand with the receptor and its action mechanism. The third chapter uses Classical and Quantum Methods to describe the conformational changes of the PfATP6 protein. The Molecular Dynamics simulations of the receptor in the isolated and complexed form with the ligands were performed for 10 ns. The final conformations obtained for the receptors were evaluated in RMSD terms, effect of the presence of ligands at the binding site and types of interactions established between the ligand and the receptor. The results showed that the PfATP6 and SERCA proteins tend to maintain their native conformations and that the model used is adequate for the purposes of the study. The monitoring of the residues of the cytoplasmic region of the proteins allowed evidencing the allosteric effect of the presence of UA and ART ligands in BS, on the A- and N-domains of PfATP6. This effect reproduces the well-established E1 and E2 conformations for PfATP6, in the presence and absence of Ca2+, respectively. Molecular Dynamics analyses corroborate the findings of Chapter II, by showing the possibility of establishing hydrogen interactions with the important residues of PfATP6 BS. These results support the evidence that Ca2+-ATPase (SERCA) pumps may be a target for the triterpenic derivatives of UA and BA.