Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Baker's yeast (Saccharomyces cerevisiae) undergoes major biochemical and structural rearrangements in order to survive cycles of desiccation and rehydration, yet a firm understanding of the response is lacking. The purpose of this study was to examine the response of S. cerevisiae to desiccation and rehydration at both the physiological and molecular levels. Transmission electron microscopy was used to show that loss of vacuolar structure, enlarged nuclear boundaries, as well as cell wall thickening were all associated with the desiccation response. Molecular analysis focused on glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.13), a multifunctional protein that is involved in several cellular processes other than glycolysis, including nuclear translocation under stress and intracellular sensing of oxidative stress during apoptosis. Here, GAPDH was studied primarily to determine its potential role in mediating the changes in cell wall physiology identified through our structural studies. GAPDH appears to be shuttled between the cell wall and the cytoplasm during the desiccation/rehydration process. Western analyses in combination with the use of inhibitors of translation (cycloheximide) suggest that the shuttling process does not require de novo protein synthesis. Western analyses also identified an immuno-reactive peptide in the cell wall and cytoplasmic fractions of lower molecular mass than native GAPDH (27 KDa vs. 37 KDa). This lower molecular weight peptide exhibited the translocation process similar to that of the full length GAPDH. Studies with GAPDH deletion strains suggested that the 27 kDa fragment is encoded by tdh3. The importance of this lower molecular weight form is yet to be determined. / Master of Science in Life Sciences

Blastocystis is an important parasite that infects humans and a wide range of animals like rats, birds, reptiles, etc. infecting a sum of 60% of world population. It belongs to the Stramenopiles, a Heterologous group that includes for example the Phythophthora infestans the responsible for the Irish potato famine. Previous work had reported the presence of an unusual fusion protein that is composed of two of the main glycolytic enzymes; Triosephosphate isomerase-glyceraldehyde-3-phosphate dehydrogenase (TPI-GAPDH). Little is known about this protein. Blastocystis TPI-GAPDH and Blastocystis enolase were both characterized biochemically and biophysically in this project. The phylogenetic relationships of those two proteins among other members of either Stramenopiles, or other members of the kingdom of life were examined and found to be grouping within the chromalveolates. Our studies revealed that those two proteins, Blastocystis enolase and Blastocystis TPI-GAPDH, had a peptide signal targeting them to the mitochondria. This was an unusual finding knowing that text books always referred to the glycolytic pathway as a canonical cytoplasmic pathway. Structural studies had also been conducted to unravel the unknown structure of the fusion protein Blastocystis TPI-GAPDH. X-ray crystallography had been conducted to solve the protein structure and the protein was found to be a tetrameric protein composed of a central tetrameric GAPDH protein flanked with two dimmers of TPI protein. Solving its structure would be the starting point towards reviling the role that TPI-GAPDH might play in Blastocystis and other organisms that it was found in as well. Although a fusion protein, the individual components of the fusion were found to contain all features deemed essential for function for TPI and GAPDH and contain all expected protein motifs for these enzymes.

Aspergillus fumigatus produces enzymes required for the hydrolysis of galactomannans like locust bean gum. Among these enzymes endo-beta-1,4 mannanase is also produced at high levels. However, the fungus is not safe for use in the food industry. Therefore, the gene encoding endo-beta-1,4-mannanase of A. fumigatus IMI 385708 was previously cloned in our laboratory into Aspergillus sojae ATCC11906 which is a safe microorganism for use in food applications. Altogether eight transformants were obtained. It was shown that some of these transformants overproduce the enzyme because of expression under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and fusion to the glucoamylase signal and pro-peptide coding region of Aspergillus niger. In this study, mannanase production of these transformants was compared with A. fumigatus and A. sojae transformant AsT1 showed c. 12 fold increase with the maximum activity of 352 U/ml. The effects of initial medium pH and number of spores on activity were investigated and maximum activity was achieved at pH 7.0 and the number of spores was found as 3.6 &times / 106. Optimization of the growth conditions for maximum mannanase production in shake flasks by using the best mannanase producing transformant AsT1 was carried out by using Box-Behnken design under Response Surface Methodology. The highest beta-mannanase activity on the fourth day of cultivation at 30 &ordm / C was obtained as 363 U/ml in the optimized medium containing 7% sugar beet molasses, 0.43% NH4NO3, 0.1% K2HPO4, 0.05% MgSO4 as the weight/volume percentage at 207 rpm. On sixth day of cultivation under the optimized conditions, the highest mannanase activity was achieved as 482 U/ml which is 1.4 fold of 352 U/ml activity found on glucose medium previously. After 48 h of LBG hydrolysis by 40 U of mannanase, mannotriose, 61-galactosyl-beta-D-mannotriose and 63,64-di-alpha-galactosyl-beta-1,4-mannopentaose were found as the main products via HPLC analysis.

In the aerobic cell, oxygen can be converted into a series of reactive metabolites, together called as "reactive oxygen species" (ROS). This large group include both radical and non-radical species such as superoxide anion (02"), hydroxyl radical ("0H), H202, nitric oxide (N0') and lipid hydroperoxides (LOOH). ROS are generated in very small amounts at all stages of aerobic life, and probably have a role in cellular regulation. However, their formation in excess leads to toxicity and damage to tissues. This situation, called 'oxidative stress', is responsible, atleast in part, to the pathophysioiogy of a number of disease states such as inflammation, arthritis, cancer, ageing, ischemia-reperfusion and several neurodegenerative disorders. Compared to other organs in the animal body, brain tissue is more vulnerable to oxidative stress. This is due to three major reasons; (1) brain has a high oxygen consumption (2) high content of polyunsaturated fatty acids and iron, that can promote lipid peroxidation, and (3) low levels of antioxidant enzymes such as catalase and glutathione peroxidase. The inability of neurons to regenerate also contributes to exacerbate an oxidant damage in the brain. The main objective of this investigation was to identify biochemical systems in the brain that are susceptible to ROS, on the following two issues: 1. What are the targets for the action of H2O2 and NO in the glycolytic cycle, the major route for the oxidation of glucose in brain? 2. What are the targets for the action of polyunsaturated fatty acids and their oxidative metabolites among the enzymes of phosphoinositide cycle (PI cycle), the ubiquitous signal transduction event in the brain? Using sheep brain cytosol , it was found that among the various glycolytic enzymes, only glyceraldehyde 3-phosphate dehydrogenase (GAPD) was inhibited by H2O2. The enzyme was purified to homogeneity from sheep brain and its inactivation with H202 was studied in detail. Commercial preparations of rabbit skeletal muscle GAPD was also used in this study. An unusual requirement of glutathione for the complete inactivtion of the enzyme by H2O2 was observed. The H2O2-inactivated GAPD was partially reactivated by prolonged treatment with thiol compounds. Using CD-spectral analysis, a significant change was found in the secondary structure in H2O2-treated GAPD. GAPD was inactivated by NO only in presence of high concentrations of DTT and after prolonged incubation. The N0-inactivated GAPD was partially reactivated by treatment with thiol compounds. A new activity, namely ADP-ribosylation (ADPR) emerged in the NO-treated mammalian, but not in yeast. GAPD, ADPR activity could be generated in GAPD through NO-independent treatments such as incubation with NADPH and aerobic dialysis. During NADPH treatment no loss of dehydrogenase activity occurred. Thus, it was concluded that loss of dehydrogenase activity and emergence of ADPR in NO-treated GAPD were not correlated but coincidental, and that NO treatment yielded small amounts of modified-GAPD that had ADPR activity. In the brain, onset of ischemia is characterized by a significant elevation in free fatty acid (FFA) levels, predominantly, arachidonic acid (AA). It is suggested that AA can be oxidised to its metabolites like prostaglandins and 15-hydroperoxy arachidonic acid (15-HPETE) and some of these might exert toxic effects during reperfusion. Using whole membranes or tissue slices prepared from rat brain, effects of polyunsaturated fatty acids and their oxidative metabolites on five enzymes of PI cycle namely PI synthase, PI and PIP kinases, agonist-stimulated PLC and DG kinase was studied. Hydroperoxides of linoleic- and arachidonic acids inactivated PI synthase selectively among the PI cycle enzymes. Interestingly, AA selectively stimulated DG kinase in neural membranes. Docasahexaenoic acid (DHA) a highly unsaturated fatty acid found in the brain, also stimulated DG kinase activity while saturated, mono-and di-unsaturated fatty acids were ineffective. It was concluded that AA and DHA have a role in modulating neural DG kinase. The data presented in the thesis indicate that ROS have selective targets in cells and the consequent protein modifications can be used to modulate cellular functions under normal and oxidative stress conditions.

No description available.

A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH, uma coleção virtual obtida do banco de dados ZINC, contendo aproximadamente 2,5 milhões de compostos, foi avaliada através de vários filtros de seleção com o objetivo de priorizar aqueles compostos mais interessantes do ponto de vista estrutural, de propriedades físico-químicas e farmacocinéticas. A aplicação desses filtros originou uma subcoleção de aproximadamente 450 mil estruturas que foram avaliadas segundo a complementaridade de interações com a estrutura da enzima através de métodos de docagem molecular. Com base nestes resultados, doze compostos que se mostraram promissores foram selecionados e adquiridos comercialmente para serem testadas in vitro contra a enzima TcGAPDH. Dos doze compostos testados, três exibiram afinidade (Ki) pela enzima em concentrações inferiores a 80 &mu;M, Além disso, esses compostos também são caracterizados pelo baixo peso molecular (274 a 330 g mol-1) e no máximo 24 átomos diferentes do hidrogênio e, como consequência, apresentam eficiências do ligante entre 0,24 e 0,34 Kcal mol-1 átomo-1, o que os tornam ótimos candidatos à otimização molecular visando aumento da afinidade pelo alvo. Para a busca de inibidores da enzima TcDHODH, primeiramente foi realizada uma busca por cavidades na estrutura 3D do alvo para a identificação de regiões distintas do sítio catalítico e passíveis de serem exploradas no planejamento de ligantes. Através desta análise foi possível o estabelecimento de quatro novas regiões com forma, volume e localizações adequadas para acomodar pequenas moléculas capazes de modular a atividade da TcDHODH. Uma destas regiões, chamada S2, localizada sob a alça &beta;4-&alpha;A e no canal de acesso dos substratos ao sítio ativo, foi escolhida para o planejamento baseado na estrutura do alvo. As estruturas de aproximadamente cem compostos derivados de pirimidinas, substituídos em posições estrategicamente definidas e selecionados através de buscas por subestruturas, foram docadas no sítio de interesse e nove compostos adquiridos e testados in vitro contra a enzima com o objetivo de validar as hipóteses estabelecidas inicialmente. Destes, cinco compostos mostraram potências (IC50) superiores à do produto de reação (inferior a 150 &mu;M), Os resultados encontrados validaram as hipóteses geradas na primeira etapa e foram usados para direcionar a seleção de outras quinze novas moléculas através de um protocolo de docagem molecular com ajuste induzido. A avaliação in vitro desses compostos contra a enzima TcDHODH resultou na identificação de outros 11 compostos ativos, dos quais o mais potente exibiu afinidade pela enzima em concentração igual a 124 nM. Este composto possui eficiência do ligante igual a 0,56 Kcal mol-1 átomo-1 e pode ser considerado um fragmento molecular com excelentes características do ponto de vista do potencial para futuro desenvolvimento como agente terapêutico. Os resultados encontrados também evidenciaram a importância de determinadas características estruturais impressas nos inibidores da TcDHODH para a complementaridade com o novo sítio de interação identificado. Assim, novos compostos foram propostos para otimização molecular com o objetivo de melhorar afinidade e aumentar a diversidade de classes e, deste modo, ampliar o espectro de perfis farmacocinéticos para posteriores ensaios celulares e in vivo, Através da realização deste trabalho foi possível validar as estratégias adotadas na utilização dos métodos computacionais e também as hipóteses construídas a partir da aplicação dos mesmos. A taxa de acerto (TA) alcançada foi superior a 30% no planejamento de inibidores para ambos os alvos, ou seja, muito superiores às encontradas em experimentos de ensaio em massa. Deste modo, este estudo contribuiu com a proposição de novos esqueletos moleculares que podem ser usados como compostos-líderes no desenvolvimento de novos agentes tripanocidas focando nas enzimas TcGAPDH e TcDHODH como alvos. / Chagas\' disease, an endemic illness widely distributed throughout Latin America, is caused by the protozoa parasite Trypanosoma cruzi. It is one of the tropical diseases that are among the most neglected by the pharmaceutical industry, for which available treatments were launched more than 30 years ago. In addition, these drugs are ineffective and cause severe side effects to patients. This panorama shows the need for the development of new and more effective chemotherapeutic agents for the treatment of the disease. Enzymes belonging to metabolic pathways that are essential for the parasite survival such as the glycolysis and pyrimidine nucleotide biosynthesis have been proposed as attractive targets for the design of new drugs for the treatment of Chagas disease. In this work, the enzymes Gyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) and the Dihydroorotate dehydrogenase (TcDHODH) from Trypanosoma cruzi were studied as targets for the design of inhibitors with physicochemical properties and structural characteristics similar to lead-compounds. Methods in Cheminformatics within the Ligand- and Structure-based Virtual Screening (LBVS and SBVS, respectively) approaches were thoroughly employed as tools to identify new hits. For the selection and identification of potential inhibitors of the GAPDH enzyme, a compound database containing nearly 2.5 million of small molecules retrievable from ZINC was evaluated through several molecular filters aiming at prioritizing those compounds more interesting from the point of view of their structures, physicochemical and predicted ADME/Tox properties. The application of Filter originated a subcollection of approximately 450 thousand structures that were then scored according to their complementary interactions with the 3D structure of the enzyme through molecular docking. Based on docking results, twelve compounds that showed to be promising ligands were selected and commercially acquired for in vitro assays against the TcGAPDH. Of the twelve compounds evaluated in vitro, three exhibited affinity constants (Ki) at concentrations lower than 80 &mu;M. Furthermore, the selected compounds are also characterized by the low molecular weight (274 to 330 g mol-1) and a maximum of non-hydrogen atom count of 24, as a result, they have Ligand Efficiencies between 0,24 and 0,34 Kcal mol-1 átomo-1, what grant them great potential as candidates for molecular optimization and potency improvement. For the search of TcDHODH inhibitors, cavities in the 3D structure of the target for the identification of areas apart from catalytic site but likely to be explored in the design of ligands were selected a priori. This resulted in four new regions with appropriate shape, volume and locations to accommodate small molecules capable of modulating the activity of TcDHODH. One of the areas, called S2 site, is located under the &alpha;4 - &beta;A loop and in the access channel of the substrate to the active site and was chosen to be explored in the SBDD studies. Approximately one hundred of pyrimidine derivatives containing strategically defined posítions for molecular substitution were retrieved from commercially available compounds database through substructure searching and docked into the previously defined site. Based on the docking results nine compounds were selected, purchased and assayed in vitro against the enzyme with the objective of validating the hypothesis. Of these, five compounds showed potencies (IC50) better than that exhibited by the product of the reaction (values lower than 150 &mu;M). Thus, the results found validated the hypotheses generated in the first stage of the designing and they were used to drive the selection of other fifteen new molecules through induced fit molecular docking protocol. The in vitro evaluation of those compounds against the TcDHODH enzyme resulted in the identification of other eleven ligands, of which the most potent exhibited affinity for enzyme at the concentration of 124 nM. This molecule has a Ligand Efficiency of 0.56 Kcal mol-1 atom-1 and can be considered a fragment-like compound with excellent characteristics from the point of view of its potential for future development as therapeutic agent. The results found also evidenced the importance of certain structural characteristics in the inhibitor of TcDHODH for the complementarily with the new identified site of interaction. Thus, new compounds were proposed for potency and class diversity improvement. By doing so we hope to enlarge ADME profile spectrum for further cellular and in vivo assays. Through the success of this work, it was possible to validate the strategies adopted in the use of computational methods and also the hypotheses built from the application of that. The success rate (TA) obtained was higher than 30% in the design of ligands for both studied targets, which is much better than that usually found along High Throughput Screening assays. Thus, this study contributed with the proposítion of new molecular scaffolds that can be used as lead compounds in the development of new tripanocidal agents having as targets the enzymes TcGAPDH or TcDHODH.

Made available in DSpace on 2017-07-20T12:40:20Z (GMT). No. of bitstreams: 1 Agnes Thiane Machado.pdf: 7176772 bytes, checksum: 01a4049f5c4aed0935803a0cf3a6468d (MD5) Previous issue date: 2017-03-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Study of protein three-dimensional structures allow us to investigate the relations between amino acid sequence, structure and function, what is important chiefly for proteins from pathogenic organisms or ones that belong to the same genus of these, such that they can be used as a structural model. In this context, this work aims at the structural characterization of the enzymes alanine racemase long isoform from Trypanosoma cruzi and glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi. The long isoform of alanine racemase catalyzes the conversion between L and D-alanine which, in turn, is part of one of the metabolic pathways in Trypanosoma cruzi, the etiologic agent of Chagas disease. The heterologous expression of this enzyme in Escherichia coli BL21 (DE3) GroEL was analyzed by SDS-PAGE, which revealed that the protein is in higher proportion in the insoluble fraction, thus it was necessary to establish a recovery protocol followed by an in vitro refolding. Data from enzymatic assays and circular dichroism revealed the success of the recovery/refolding protocol, which may in the future contribute to the search for specific inhibitors. Glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi catalyzes the sixth step of the organism’s glycolytic pathway. NgGAPDH enzyme was expressed in E. coli (DE3) using the pET-15b vector, and then purified by tree chromatographic steps, two of nickel affinity and one of size exclusion. The enzymatic characterization was investigated with the enzyme without the his-tag; NgGAPDH presented higher activity at pH 8.0, 25 °C and 10 mM of arsenate, and positive cooperativity for substrates G3P and NAD+. His-tag depleted NgGAPDH crystals appeared in 3 days after drop settings, the best crystal diffracted to 1.94 A resolution and belongs to space group P21 with cell parameters a = 83.74 A, b = 94.55 A, c = 90.93 A, = 99.96 °. The final refined structure presents R = 0.1652 and Rfree = 0.2029. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motion with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". It should be emphasized that this is the first GAPDH from the phylum Percolozoa that has its three dimensional structure determined and kinetic parameters established, such that we expect to have contributed to the understanding of the evolution of this class of proteins. / O estudo da estrutura tridimensional de proteínas nos permite investigar as relações entre sequência de aminoácidos, estrutura e função, o que é importante principalmente para proteínas de organismos patogênicos ou mesmo pertencente ao gênero destes, que podem ser utilizadas como modelo estrutural. Neste contexto, o presente trabalho visa caracterizar estruturalmente as enzimas alanina racemase isoforma longa de Trypanosoma cruzi e gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi. A alanina racemase isoforma longa catalisa a conversão entre L e D-alanina em uma das vias metabólicas do T. cruzi, que é o agente etiológico da doença de Chagas. A análise por SDS-PAGE de amostras da expressão heteróloga dessa enzima em Escherichia coli BL21(DE3) GroEL revelou que a proteína está em maior proporção na fração insolúvel, por isso, foi necessário estabelecer um protocolo de recuperação seguido de um reenovelamento in vitro. Dados de ensaios enzimáticos e dicroísmo circular revelaram o sucesso do protocolo de recuperação/reenovelamento, o que poderá no futuro contribuir para a busca de inibidores específicos. A gliceraldeído-3-fosfato desidrogenase de Naegleria gruberi catalisa a sexta etapa da via glicolítica do organismo. A enzima NgGAPDH foi expressa em E. coli (DE3) usando-se o vetor pET-15b e então purificada em três passos de cromatografia, dois por afinidade a níquel e um por exclusão por tamanho. A caracterização enzimática foi realizada com a enzima sem a ―his-tag‖; a NgGAPDH apresentou maior atividade em pH 8,0, 25 °C e 10 mM de arsenato, e cooperatividade positiva frente aos substratos G3P e NAD+. Cristais de NgGAPDH sem a ―his-tag‖ apareceram em 3 dias após montagem das gotas e o melhor difratou a 1,94 A de resolução, pertencendo ao grupo espacial P21 com parâmetros de cela a = 83,74 Å, b = 94,55 A, c = 90,93 A e = 99,96 °. A estrutura final refinada apresenta R = 0,1652 e Rfree = 0,2029. O domínio catalítico formado pelos resíduos 134 a 313 é altamente conservado, como esperado, com exceção da Asn145, presente somente em NgGAPDH, enquanto que as demais GAPDHs apresentam Ser ou Thr na posição correspondente. Análises por dinâmica molecular revelaram que a Asn145 tem correlação de movimento com os resíduos Ala123, Thr125 e Pro126, pertencentes ao que se chamou de ―bonded loop‖. Ressalte-se que esta é a primeira GAPDH do filo Percolozoa que tem sua estrutura tridimensional determinada e parâmetros cinéticos estabelecidos, tal que se espera contribuir para o entendimento da evolução dessa classe de proteínas.

A Doença de Chagas, causada pelo parasito tripanossomatídeo Trypanosoma cruzi, é endêmica e se distribuí por toda América Latina. É uma das parasitoses mais negligenciadas pela indústria farmacêutica e os únicos fármacos disponíveis para seu tratamento foram introduzidos há décadas. Infelizmente, eles são ineficientes e apresentam sérios efeitos colaterais. Esse panorama mostra a necessidade do desenvolvimento de novos fármacos para a quimioterapia contra a doença de Chagas. As enzimas pertencentes a vias metabólicas essenciais para a sobrevivência do parasito tais como a via glicolítica e a de síntese de novo de nucleotídeos de pirimidinas, têm sido propostas como alvos interessantes no planejamento novos fármacos para o tratamento da doença de Chagas. Neste trabalho, as enzimas Gliceraldeído 3-fosfato desidrogenase (TcGAPDH) e a Diidroorotato desidrogenase (TcDHODH) de Trypanosoma cruzi foram estudadas como alvos para o planejamento de inibidores enzimáticos com propriedades físico-químicas e características estruturais similares à de compostos-líderes. Para isso, foram utilizados métodos e ferramentas de Quiminformática tanto baseadas nas estruturas dos ligantes (LBVS) quanto dos receptores (SBVS). Para a identificação e seleção de potenciais inibidores da enzima GAPDH, uma coleção virtual obtida do banco de dados ZINC, contendo aproximadamente 2,5 milhões de compostos, foi avaliada através de vários filtros de seleção com o objetivo de priorizar aqueles compostos mais interessantes do ponto de vista estrutural, de propriedades físico-químicas e farmacocinéticas. A aplicação desses filtros originou uma subcoleção de aproximadamente 450 mil estruturas que foram avaliadas segundo a complementaridade de interações com a estrutura da enzima através de métodos de docagem molecular. Com base nestes resultados, doze compostos que se mostraram promissores foram selecionados e adquiridos comercialmente para serem testadas in vitro contra a enzima TcGAPDH. Dos doze compostos testados, três exibiram afinidade (Ki) pela enzima em concentrações inferiores a 80 &mu;M, Além disso, esses compostos também são caracterizados pelo baixo peso molecular (274 a 330 g mol-1) e no máximo 24 átomos diferentes do hidrogênio e, como consequência, apresentam eficiências do ligante entre 0,24 e 0,34 Kcal mol-1 átomo-1, o que os tornam ótimos candidatos à otimização molecular visando aumento da afinidade pelo alvo. Para a busca de inibidores da enzima TcDHODH, primeiramente foi realizada uma busca por cavidades na estrutura 3D do alvo para a identificação de regiões distintas do sítio catalítico e passíveis de serem exploradas no planejamento de ligantes. Através desta análise foi possível o estabelecimento de quatro novas regiões com forma, volume e localizações adequadas para acomodar pequenas moléculas capazes de modular a atividade da TcDHODH. Uma destas regiões, chamada S2, localizada sob a alça &beta;4-&alpha;A e no canal de acesso dos substratos ao sítio ativo, foi escolhida para o planejamento baseado na estrutura do alvo. As estruturas de aproximadamente cem compostos derivados de pirimidinas, substituídos em posições estrategicamente definidas e selecionados através de buscas por subestruturas, foram docadas no sítio de interesse e nove compostos adquiridos e testados in vitro contra a enzima com o objetivo de validar as hipóteses estabelecidas inicialmente. Destes, cinco compostos mostraram potências (IC50) superiores à do produto de reação (inferior a 150 &mu;M), Os resultados encontrados validaram as hipóteses geradas na primeira etapa e foram usados para direcionar a seleção de outras quinze novas moléculas através de um protocolo de docagem molecular com ajuste induzido. A avaliação in vitro desses compostos contra a enzima TcDHODH resultou na identificação de outros 11 compostos ativos, dos quais o mais potente exibiu afinidade pela enzima em concentração igual a 124 nM. Este composto possui eficiência do ligante igual a 0,56 Kcal mol-1 átomo-1 e pode ser considerado um fragmento molecular com excelentes características do ponto de vista do potencial para futuro desenvolvimento como agente terapêutico. Os resultados encontrados também evidenciaram a importância de determinadas características estruturais impressas nos inibidores da TcDHODH para a complementaridade com o novo sítio de interação identificado. Assim, novos compostos foram propostos para otimização molecular com o objetivo de melhorar afinidade e aumentar a diversidade de classes e, deste modo, ampliar o espectro de perfis farmacocinéticos para posteriores ensaios celulares e in vivo, Através da realização deste trabalho foi possível validar as estratégias adotadas na utilização dos métodos computacionais e também as hipóteses construídas a partir da aplicação dos mesmos. A taxa de acerto (TA) alcançada foi superior a 30% no planejamento de inibidores para ambos os alvos, ou seja, muito superiores às encontradas em experimentos de ensaio em massa. Deste modo, este estudo contribuiu com a proposição de novos esqueletos moleculares que podem ser usados como compostos-líderes no desenvolvimento de novos agentes tripanocidas focando nas enzimas TcGAPDH e TcDHODH como alvos. / Chagas\' disease, an endemic illness widely distributed throughout Latin America, is caused by the protozoa parasite Trypanosoma cruzi. It is one of the tropical diseases that are among the most neglected by the pharmaceutical industry, for which available treatments were launched more than 30 years ago. In addition, these drugs are ineffective and cause severe side effects to patients. This panorama shows the need for the development of new and more effective chemotherapeutic agents for the treatment of the disease. Enzymes belonging to metabolic pathways that are essential for the parasite survival such as the glycolysis and pyrimidine nucleotide biosynthesis have been proposed as attractive targets for the design of new drugs for the treatment of Chagas disease. In this work, the enzymes Gyceraldehyde-3-phosphate dehydrogenase (TcGAPDH) and the Dihydroorotate dehydrogenase (TcDHODH) from Trypanosoma cruzi were studied as targets for the design of inhibitors with physicochemical properties and structural characteristics similar to lead-compounds. Methods in Cheminformatics within the Ligand- and Structure-based Virtual Screening (LBVS and SBVS, respectively) approaches were thoroughly employed as tools to identify new hits. For the selection and identification of potential inhibitors of the GAPDH enzyme, a compound database containing nearly 2.5 million of small molecules retrievable from ZINC was evaluated through several molecular filters aiming at prioritizing those compounds more interesting from the point of view of their structures, physicochemical and predicted ADME/Tox properties. The application of Filter originated a subcollection of approximately 450 thousand structures that were then scored according to their complementary interactions with the 3D structure of the enzyme through molecular docking. Based on docking results, twelve compounds that showed to be promising ligands were selected and commercially acquired for in vitro assays against the TcGAPDH. Of the twelve compounds evaluated in vitro, three exhibited affinity constants (Ki) at concentrations lower than 80 &mu;M. Furthermore, the selected compounds are also characterized by the low molecular weight (274 to 330 g mol-1) and a maximum of non-hydrogen atom count of 24, as a result, they have Ligand Efficiencies between 0,24 and 0,34 Kcal mol-1 átomo-1, what grant them great potential as candidates for molecular optimization and potency improvement. For the search of TcDHODH inhibitors, cavities in the 3D structure of the target for the identification of areas apart from catalytic site but likely to be explored in the design of ligands were selected a priori. This resulted in four new regions with appropriate shape, volume and locations to accommodate small molecules capable of modulating the activity of TcDHODH. One of the areas, called S2 site, is located under the &alpha;4 - &beta;A loop and in the access channel of the substrate to the active site and was chosen to be explored in the SBDD studies. Approximately one hundred of pyrimidine derivatives containing strategically defined posítions for molecular substitution were retrieved from commercially available compounds database through substructure searching and docked into the previously defined site. Based on the docking results nine compounds were selected, purchased and assayed in vitro against the enzyme with the objective of validating the hypothesis. Of these, five compounds showed potencies (IC50) better than that exhibited by the product of the reaction (values lower than 150 &mu;M). Thus, the results found validated the hypotheses generated in the first stage of the designing and they were used to drive the selection of other fifteen new molecules through induced fit molecular docking protocol. The in vitro evaluation of those compounds against the TcDHODH enzyme resulted in the identification of other eleven ligands, of which the most potent exhibited affinity for enzyme at the concentration of 124 nM. This molecule has a Ligand Efficiency of 0.56 Kcal mol-1 atom-1 and can be considered a fragment-like compound with excellent characteristics from the point of view of its potential for future development as therapeutic agent. The results found also evidenced the importance of certain structural characteristics in the inhibitor of TcDHODH for the complementarily with the new identified site of interaction. Thus, new compounds were proposed for potency and class diversity improvement. By doing so we hope to enlarge ADME profile spectrum for further cellular and in vivo assays. Through the success of this work, it was possible to validate the strategies adopted in the use of computational methods and also the hypotheses built from the application of that. The success rate (TA) obtained was higher than 30% in the design of ligands for both studied targets, which is much better than that usually found along High Throughput Screening assays. Thus, this study contributed with the proposítion of new molecular scaffolds that can be used as lead compounds in the development of new tripanocidal agents having as targets the enzymes TcGAPDH or TcDHODH.