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Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni / Biochemical, biophysical and inhibitory studies of dihydroorotate dehydrogenase from Schistosoma mansoniJuliana Serafim David Costacurta 26 September 2014 (has links)
Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.
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Caracterização bioquímica, biofísica e estudos inibitórios da enzima diidroorotato desidrogenase de Schistosoma mansoni / Biochemical, biophysical and inhibitory studies of dihydroorotate dehydrogenase from Schistosoma mansoniCostacurta, Juliana Serafim David 26 September 2014 (has links)
Muitas doenças parasitárias, consideradas negligenciadas devido à falta de investimentos para o desenvolvimento de novas estratégias de prevenção e tratamento por parte dos setores público e privado, constituem um grave problema de saúde pública mundial e um obstáculo ao desenvolvimento sócio-econômico de países pobres e emergentes. A esquistossomose, em especial, é uma parasitose causada por platelmintos trematódeos do gênero Schistosoma que afeta 78 países e aproximadamente 249 milhões de pessoas. No Brasil, o S. mansoni é o agente etiológico causador da esquistossomose, chega a atingir 19 estados e aproximadamente 6 milhões de indivíduos. Embora atualmente o fármaco praziquantel seja utilizado para o tratamento da esquistossomose, há a necessidade de busca por novas opções terapêuticas, uma vez que este possui eficácia restrita ao estágio adulto do parasita, efeitos colaterais que dificultam a adesão do paciente ao tratamento e, dada a massiva administração do medicamento, a resistência do parasita ao medicamento pode se tornar um sério problema de saúde pública. Dentro deste contexto, existe um grande interesse em buscar novos alvos macromoleculares e em particular investigar o potencial da enzima diidroorotato desidrogenase (DHODH) como possível alvo terapêutico para o desenvolvimento de terapias eficazes e seguras para o tratamento da esquistossomose. A enzima DHODH participa da quarta etapa enzimática da via de biossíntese de nucleotídeos pirimidínicos, e estudos recentes demonstram que a inibição específica desta enzima compromete a produção de nucleotídeos, e consequentemente a proliferação celular. Na verdade a enzima DHODH já é alvo validado para o tratamento de doenças como o câncer, a artrite reumatoide e doenças parasitárias como a malária. Como primeira etapa para a avaliação do potencial terapêutico da enzima DHODH no tratamento da esquistossomose, este projeto propõe a caracterização bioquímica e biofísica da DHODH de Schistosoma mansoni, bem como a identificação de inibidores para esta enzima. Os resultados obtidos até o presente momento consistem no desenvolvimento de um protocolo de expressão e purificação que permitiram a obtenção de proteína pura e com rendimento de 40 miligramas de proteína por litro de meio de cultura. Nossos estudos demonstraram que a proteína se mostra mais estável na presença de detergente, alta concentração de sal e glicerol. Ensaios de espalhamento dinâmico de luz realizados a partir de amostras de SmDHODH purificadas a partir da associação de cromatografia por afinidade com cromatografia por exclusão molecular foram utilizados para a caracterização de uma população homogênea de diâmetro aproximado de 90 Å. Ensaios de atividade enzimática e de inibição foram realizados para SmDHODH, como também para a proteína homóloga humana, HsDHODH, de forma a permitir estudos comparativos. Os resultados sugerem que o pH ótimo da reação para ambas as enzimas se encontra na faixa entre 8,0 e 8,5. O protocolo de caracterização cinética desenvolvido para estas enzimas permitiu a obtenção dos parâmetros KM e kcat, assim como dar início à realização de ensaios de inibição na presença de bancos de ligantes de origem sintética e natural. Os resultados cinéticos obtidos sugerem que a SmDHODH e a HsDHODH seguem o mecanismo ii Ping-Pong, de acordo com o que já foi descrito para as outras DHODHs, com os seguintes valores de KM e kcat: KDHO= 174 ± 18 ?M; KQo= 159 ± 18 ?M; e kcat= 27 ± 1 s-1 para a SmDHODH e KDHO= 286 ± 31 ?M; KQo= 354 ± 38 ?M; e kcat= 78 ± 4 s-1 para a HsDHODH. Foram identificados compostos químicos com potencial inibitório na faixa de 794 ? 3 ?M a 19,1 ? 0,1 nM para a SmDHODH e de 33,9 ? 0,1 ?M a 37,2 ? 0,1 nM para a HsDHODH. Os resultados deste trabalho aliado aos estudos estruturais em desenvolvimento pelo nosso laboratório serão utilizados não só para a completa caracterização da enzima, mas também para o futuro planejamento de ligantes específicos baseados na estrutura e função protéica, como uma importante ferramenta no combate à esquistossomose. / Many parasitic diseases, considered neglected due to lack of investment from the public and private sectors in the development of new strategies for prevention and treatment, are a serious global public health problem and a hindrance to the development of poor and emergent countries. Schistosomiasis, in particular, is a parasitic disease caused by trematode plathelmintes of the genus Schistosoma that affects 78 countries and approximately 249 million people. In Brazil, S. mansoni, the endemic etiologic agent of schistosomiasis, is found in 19 states and affects approximately 6 million people. Although the drug praziquantel is currently used for the treatment of schistosomiasis, this drug has limited effectiveness in the adult stage of the parasite, many side effects hamper the adherence to the patient´s treatment and, given the intense drug usage, resistant parasites can, very soon, become a serious public health problem. Thus, there is a real need for the search of new therapeutic options. Within this context, there is a great interest in the search for new macromolecular targets against Schistosoma mansoni and in particular, to investigate the enzyme dihydroorotate dehydrogenase (DHODH) as new therapeutic target for the treatment of schistosomiasis. DHODH catalyzes the conversion of dihydroorotate (DHO) to orotate (ORO) in the fourth step of the pyrimidine nucleotides pathway. Recent studies show that specific inhibition of this enzyme commits nucleotides biosynthesis and, consequently, cell proliferation. DHODH is, in fact, a validated target for the treatment of diseases such as cancer, rheumatoid arthritis and malaria. As a first step towards the evaluation of the therapeutic potential of DHODH from S. mansoni (SmDHODH) for the treatment of schistosomiasis, this project proposed the biochemical and biophysical characterization, as well as the identification of inhibitors for this enzyme. The results obtained so far included the development of an expression and purification protocol that allowed us to obtain pure protein with a good yield. In addition, our findings reveals that for SmDHODH stabilization the enzyme requires a buffer containing detergent, glycerol and high salt concentration. Dynamic light scattering studies performed with SmDHODH protein samples purified by a combination of both affinity chromatography and size exclusion chromatography allowed the characterization of a homogeneous population with approximately 90 Å diameter. In order to allow comparative studies, enzymatic and inhibitory assays were performed for SmDHODH as well as for the human homologous enzyme (HsDHODH). The results suggest that for both enzymes the optimum pH for the enzymatic reaction is found in the range of 8.0 and 8.5. The enzymatic assay developed for this class of enzymes allowed the characterization of the kinetic parameters KM and kcat for both enzymes, as well as the performance of inhibitory assays in the presence of synthetic and natural ligands. The inhibition tests allowed us the identification of chemical compounds that inhibit SmDHODH in the range of 794 ? 3 ?M to 19.1 ? 0.1 nM and HsDHODH in the range of 33.9 ? 0.1 ?M a 37.2 ? 0.1 nM. The results of this work, together with structural studies currently in progress in our laboratory will be exploited for the complete characterization of the iv enzyme, as well as for the development of specific inhibitors of SmDHODH, as an important tool in the fight against schistosomiasis.
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Structural Studies of the Transmembrane and Membrane Proximal Domains of HIV-1 gp41 by X-Ray CrystallographyJanuary 2014 (has links)
abstract: The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2014
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SYNTHESIS AND DEVELOPMENT OF ZWITTERIONIC PEI (zPEI) FOR OPTIMIZED DELIVERY OF NUCLEIC ACIDSDuke, Joseph Raleigh, III 01 January 2017 (has links)
Gene therapy holds promise for the treatment a wide range of diseases ranging from cystic fibrosis to cardiovascular disease to cancer. The need for safe and efficient gene delivery methods remains the primary barrier to human gene therapy. Non-viral vector materials, including polymers, can be designed to be biocompatible and non-immunogenic, but lack the efficiency to be clinically relevant. Gene therapy awaits the development of new materials that are both safe and efficient. Here, we have synthesized a series of modified zwitterionic polymers based on the common transfecting agent polyethylenimine (PEI). Using a variety of biochemical and biophysical methods we have studied structure-function relation in zPEI-DNA as a function of percent modification. Our results show significant structural rearrangements in the DNA condensates with increasing zwitterionic character. The percent zwitterionic modification determines not only DNA packaging but the serum stability of the resulting polyplexes with more highly modified zPEI releasing DNA more readily.
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Overexpression and structure-function characterization of HIV-1 Subtype C. reverse transcriptase and proteaseTambani, Tshifhiwa 20 September 2019 (has links)
PhD (Microbiology) / Department of Microbiology / High genetic diversity is a major contributory factor in the development of drug resistance, in
addition to challenges in diagnosis and treatment monitoring in the therapeutics of human
immunodeficiency virus (HIV) .Within the wide HIV-1 diversity, differences in mutational
frequency, disease progression, drug response and transmission amongst HIV-1 subtypes
have been shown. In spite HIV-1 subtype C (HIV-1C) being the most prevalent variant globally,
none of the available drugs nor screening assays for inhibitory molecules have been developed
targeting the genetics of this important subtype. This study therefore aimed to overexpress and
biophysically characterize HIV-1C reverse transcriptase and protease to serve as reagents in
the development of assays for routine screening of molecules inhibitory to HIV-1C.
Heterologous expression of HIV-1C reverse transcriptase and protease isolates that are
prevalent in South Africa was carried out in Escherichia coli (E. coli (BL21-DE3). The secondary
and tertiary structures of the proteins were determined using, circular dichroism (CD) and
fluorescence spectroscopy respectively. Thereafter, interaction studies to delineate interaction
properties of natural products for possible inhibition of protease were conducted. Furthermore,
in silico studies to determine binding interactions, further confirmed by in vitro binding assays
of a pepsin inhibitor homolog (Bm-33) from Brugia malayi , against protease were also
conducted. Expressed reverse transcriptase and protease from the globally prevalent HIV-1C
were shown to be structurally and functionally intact for application in downstream HIV-1
inhibition assays. Interaction studies on the other hand revealed successful inhibition of the
expressed HIV-1C PR with gallotanin. Furthermore, binding interactions of Bm-33 and HIV-1
PR revealed the first intermolecular interactions of the two molecules displaying possible
inhibition of HIV-1 PR / NRF
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Charakterizace rekombinantního fragmentu protilátky proti znaku CD3 / Characterization of recombinant fragment of an antibody against CD3 marker.Písačková, Jana January 2011 (has links)
Monoclonal antibody MEM-57 recognizes CD3 antigen expressed on peripheral blood T-lymphocytes. CD3 surface glycoprotein complex associates with T-cell receptor and is responsible for the transduction of activation signal. Antibody MEM-57 has, therefore, a large diagnostic and therapeutic potential. It could be used in autoimmune diseases diagnostics, for classification of T-cell leukemias and, as an immunosuppressant, in transplantation. The most promising therapeutic use of MEM-57 antibody would be the construction of a "Bispecific T-cell Engager" (BiTE) antibody format with potential application in cancer therapy. In this format, single-chain variable fragment (scFv) of MEM-57 would be fused with an anti-tumor antigen scFv. The thesis is focused on biochemical and biophysical characterization of MEM-57 antibody scFv fragment. Recombinant antibody fragment scFv MEM-57, equipped with the pelB leader sequence, c-myc tag and His5 tag, was produced from a pET22b(+) vector into the periplasmic space of E. coli BL21 (DE3). Two-step purification protocol, employing nickel chelation affinity chromatography and ion-exchange chromatography, was developed to obtain high yield of pure protein. The antigen binding activity of scFv MEM-57 was confirmed by flow cytometry. Structural information on scFv MEM-57...
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