<p>This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium <i>Finegoldia magna</i>. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule.</p><p>Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.</p>

This thesis describes the crystal structures of two proteins in the context of combatting bacterial infections. The GA module is a bacterial albumin-binding domain from a surface protein expressed by pathogenic strains of the human commensal bacterium Finegoldia magna. The structure of the GA module in complex with human serum albumin (HSA) provides insights into bacterial immune evasion, where pathogenicity is acquired by the bacterial cell through the ability to coat (and disguise) itself with serum proteins. The structure shows binding of the GA module to HSA in the presence of fatty acids, and reveals interactions responsible for the host range specificity of the invading bacterium. The complex resulting from binding of the GA module to HSA readily forms stable crystals that permit structural studies of drug binding to HSA. This was exploited to study the specific binding of the drug naproxen to the albumin molecule. Antibiotics play a major role in controlling infections by attacking invading bacteria. The enzyme deacetylcephalosporin C acetyltransferase (DAC-AT) catalyses the last step in the biosynthesis of the beta-lactam antibiotic cephalosporin C, one of the clinically most important antibiotics in current use. The enzyme uses acetyl coenzyme A as cofactor to acetylate a biosynthetic intermediate. Structures of DAC-AT in complexes with reaction intermediates have been determined. The structures suggest that the acetyl transfer reaction proceeds through a double displacement mechanism, with acetylation of a catalytic serine by the cofactor through a suggested tetrahedral transition state, followed by acetyl transfer to the intermediate through a second suggested tetrahedral transition state. The structure of DAC-AT yields valuable information for the continued study of cephalosporin biosynthesis in the context of developing new beta-lactam compounds.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.

Neste trabalho, foram desenvolvidos e validados métodos qualitativos e quantitativos para o controle de qualidade de cefpiroma, antibiótico β-lactâmico de amplo espectro utilizado, principalmente, no tratamento de infecções graves e episódios febris em pacientes com neutropenia, na forma farmacêutica pó para solução injetável. A substância química de referência utilizada nas análises foi caracterizada por espectrofotometria no infravermelho e por espectroscopia de ressonância magnética nuclear. A análise qualitativa foi realizada por cromatografia em camada delgada, determinação da faixa de fusão, espectrofotometria nas regiões do ultravioleta e do infravermelho e cromatografia líquida de alta eficiência, possibilitando a identificação da amostra. A determinação quantitativa foi realizada através dos métodos: espectrofotometria no ultravioleta, cromatografia líquida de alta eficiência e ensaio microbiológico pelo método de difusão em ágar – cilindros em placas, delineamento 3 x 3. Os métodos propostos foram validados segundo guias oficiais e demonstraram ser específicos, lineares, precisos e exatos. Os resultados obtidos demonstraram não haver diferença significativa entre os métodos quando comparados estatisticamente pela análise de variância (ANOVA). Outro objetivo deste trabalho foi o estudo da estabilidade da cefpiroma na forma farmacêutica reconstituída com água para injetável. Os fatores de degradação avaliados foram temperatura (40 ºC) e luz (UVC – 254 nm). Os estudos acelerados de estabilidade demonstraram que a cinética de degradação térmica e fotoquímica foi de primeira ordem. Um produto de degradação térmica foi isolado e identificado, por ressonância magnética nuclear, espectrofometria no infravermelho e espectroscopia de massa, como sulfato de 6-7-diidro-5H-ciclopenta[b]piridinium. / The aim of this study was the development and validation of analytical methods to the determination of cefpirome in powder for injectable preparation and the stability study of the drug after reconstitution of the pharmaceutical dosage form with injectable water. Cefpirome is a fourth-generation cephalosporin active against a broad spectrum of gram-negative and gram-positive bacterial infections and its principal indication is in the treatment of patients’ septic shock or several sepsis. The substance used as reference standard in the analysis was characterized by infrared spectroscopy and nuclear magnetic resonance. The qualitative analysis was performed by thin layer chromatography, melting range determination, infrared and ultraviolet spectroscopy and high performance liquid chromatography, allowing the identification of samples. The quantitative determination was performed by ultraviolet spectrophotometry, high performance liquid chromatography and cylinder-plate microbiological assay (3 x 3). The proposed methods were validated following official guides and all of them demonstrated to be specific, linear, precise and accurate. The obtained results were analyzed by the ANOVA method and they are not statistically different. The degradation factors evaluated were the temperature (40 ºC) and light (UVC – 254 nm) and the accelerated stability demonstrated that the degradation kinetics from thermal and photo degradation were first-order reactions. A thermal degradation product was isolated and identified, by nuclear magnetic resonance, infrared spectroscopy and mass spectrometry, as 6-7-dihidro-5Hcyclopenta[ b]pyridinium sulfate.