O envenenamento ofídico é considerado uma condição tropical negligenciada pela OMS, e no Brasil, o gênero Bothrops está envolvido na maioria dos casos. Primeiramente, estudamos a atividade hidrolítica do veneno de B. jararaca sobre peptídeos biologicamente ativos que podem estar relacionadas com o envenenamento. A hidrólise dos peptídeos que foram substratos para as serinopeptidases não foi eficientemente bloqueada pelo soro antibotrópico produzido pelo Instituto Butantan e, portanto, as causas dessas falhas foram investigadas. Para isso, purificamos quatro serinopeptidases não bloqueadas pelo soro e, por estudos imunoquímicos, observamos que apesar deste não bloquear as atividades destas enzimas, o mesmo é capaz de reconhecê-las. Portanto, decidimos obter soros experimentais contra estas moléculas utilizando camundongos, a fim de compará-los com o soro comercial. Os soros experimentais contra as serinopeptidases mostraram capacidade de reconhecimento e alta afinidade contra elas, e mais importante, capacidade de neutralizar suas atividades in vitro. / Snakebite is considered a neglected tropical condition by WHO, and in Brazil, the Bothrops genus is involved in most of the cases. Initially, we have studied the B. jararaca venom activity over bioactive peptides which could be related with the envenomation. The hydrolysis of the peptides substrate for serinepeptidases were not efficiently blocked by the Butantan Institute bothropic antivenom, therefore, the causes of this flaw were investigated. Thereafter, we purified four serinepeptidases not blocked by the antivenom and, by immunochemistry analysis, we observed that although it could not neutralize the activity, it could well recognize these proteins. Thus, we decided to obtain experimental sera against these serinepeptidases in mice, in order to compare it with the commercial antivenom. The experimental sera against these enzymes demonstrated recognition capability and high affinity, and most important, the ability to neutralize their activity in vitro.

IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...

Visceral adipose tissue derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that  were specifically expressed or overexpressed in the intra abdominal or visceral adipose tissue  (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral  obesity, insulin resistance, hyperinsulinemia and ‐glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type  2 diabetes.  The follow-up study reporting the cloning, expression and functional characterization of  vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.

Résumé: Chaque année, les épidémies saisonnières d’influenza causent de 3 à 5 millions de cas sévères de maladie, entraînant entre 250 000 et 500 000 décès mondialement. Seulement deux classes d’antiviraux sont actuellement commercialisées pour traiter cette infection respiratoire : les inhibiteurs de la neuraminidase, tels que l’oseltamivir (Tamiflu) et les inhibiteurs du canal ionique M2 (adamantanes). Toutefois, leur utilisation est limitée par l’apparition rapide de résistance virale. Il est donc d’un grand intérêt de développer de nouvelles stratégies thérapeutiques pour le traitement de l’influenza. Le virus influenza dépend de l’activation de sa protéine de surface hémagglutinine (HA) pour être infectieux. L’activation a lieu par clivage protéolytique au sein d’une séquence d’acides aminés conservée. Ce clivage doit être effectué par une enzyme de l’hôte, étant donné que le génome du virus ne code pour aucune protéase. Pour les virus infectant l’humain, plusieurs études ont montré le potentiel de protéases à sérine transmembranaires de type II (TTSP) à promouvoir la réplication virale : TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 et matriptase, identifiée récemment par notre équipe (Beaulieu, Gravel et al., 2013), activent l’HA des virus influenza A (principalement H1N1 et H3N2). Toutefois, il existe peu d’information sur le clivage de l’HA des virus influenza B, et seulement TMPRSS2 et HAT ont été identifiées comme étant capables d’activer ce type de virus. Les travaux de ce projet de maîtrise visaient à identifier d’autres TTSP pouvant activer l’HA de l’influenza B. L’efficacité de clivage par la matriptase, hepsine, HAT et Desc1 a été étudiée et comparée entre ces TTSP. Ces quatre protéases s’avèrent capables de cliver l’HA de l’influenza B in vitro. Cependant, seul le clivage par matriptase, hepsine et HAT promeut la réplication virale. De plus, ces TTSP peuvent aussi supporter la réplication de virus influenza A. Ainsi, l’utilisation d’un inhibiteur de TTSP, développé en collaboration avec notre laboratoire, permet de bloquer significativement la réplication virale dans les cellules épithéliales bronchiques humaines Calu-3. Cet inhibiteur se lie de façon covalente et lentement réversible au site actif de la TTSP par un mécanisme slow tight-binding. Puisque cet inhibiteur cible une composante de la cellule hôte, et non une protéine virale, il n’entraîne pas le développement de résistance après 15 passages des virus en présence de l’inhibiteur dans les cellules Calu-3. L’inhibition des TTSP activatrices d’HA dans le système respiratoire humain représente donc une nouvelle stratégie thérapeutique pouvant mener au développement d’antiviraux efficaces contre l’influenza. / Abstract: Seasonal influenza epidemics cause between 3 and 5 millions severe cases of disease, leading to 250 000 to 500 000 deaths worldwide. Only two classes of drugs are currently available to treat influenza infections: neuraminidase inhibitors, such as oseltamivir (Tamiflu) and M2 channel inhibitors (adamantanes). However, the use of these antivirals is restricted by rapid emergence of viral resistance. It is therefore of great interest to develop new therapeutic strategies for the treatment of influenza disease. The influenza virus requires activation of its surface protein hemagglutinin (HA) to become infectious. This activation is achieved by proteolytic cleavage in a highly conserved amino acid sequence of the protein. Host cell proteases are responsible for this cleavage since the viral genome doesn’t encode any protease. For viruses that infect humans, many studies have shown the potential of type II transmembrane serine proteases (TTSP) to promote viral replication: TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 and matriptase, recently identified by our team (Beaulieu, Gravel et al., 2013), activate HA of influenza A viruses (mainly H1N1 and H3N2). However, little is known about cleavage of influenza B virus HA, and only TMPRSS2 and HAT have been identified as being capable of activating this type of virus. This project aimed to identify other TTSPs able to activate influenza B HA. Cleavage efficacies of matriptase, hepsin, HAT and Desc1 were studied and compared. These four proteases were shown to be able to cleave influenza B HA using in vitro assays. However, only cleavage by matriptase, hepsin and HAT promoted viral replication. Moreover, these TTSPs also supported the replication of influenza A viruses. Thus, the use of a slow, tight-binding inhibitor (developed in collaboration with our laboratory) that binds to the TTSP active site, forming a covalent and reversible bond, significantly blocked viral replication in human bronchial epithelial Calu-3 cells. Since this inhibitor targets a host cell component, instead of a viral protein, viruses did not develop resistance after 15 passages in presence of the inhibitor in Calu-3 cells. Thus, inhibition of HA-activating TTSPs in the human respiratory tract represents a novel therapeutic strategy against influenza.

INTRODUÇÃO: A asma ainda acomete um número crescente de indivíduos, podendo ser muito grave e, algumas vezes, fatal. A despeito da melhor eficiência diagnóstica e eficácia terapêutica, a maioria dos asmáticos graves não obtém controle total dos sintomas com as terapias disponíveis. Alguns estudos sugerem a atuação de inibidores de serinoproteases em diversos processos inflamatórios, entre estes inibidores encontra-se o Boophilus microplus trypsin Inhibitor (BmTI-A). OBJETIVO: Avaliar se o recombinante do inibidor de serinoproteases rBmTI-A modula a hiperresponsividade brônquica à metacolina, inflamação e remodelamento das vias aéreas em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Camundongos Balb-c foram divididos em 4 grupos: SAL (salina), OVA (sensibilizados com ovoalbumina), SAL+rBmTI-A (controle tratados com rBmTI-A ) e OVA+rBmTI-A (sensibilizados com ovoalbumina e tratados com rBmTI-A). Nos dias 0 e 14 do protocolo, os animais receberam injeção intraperitoneal (i.p) de salina (0,9% NaCl) (SAL e SAL+rBmTI-A) e ovoalbumina (50 ug/mL) (OVA e OVA+rBmTI-A). Nos dias 22, 24, 26 e 28 foram submetidos à inalação com salina (0,9% NaCl) ou ovoalbumina (10 mg/ml) e foram tratados com rBmTI-A (35,54 pmol em 50 uL de NaCl) ou apenas salina, via instilação nasal, nos dias 22 e 28. No dia 29, foram realizadas as seguintes análises: hiper-responsividade à metacolina e respostas máximas de resistência e elastância do sistema respiratório; quantificação do número total de células, macrófagos, linfócitos e polimorfonucleares no fluido do lavado broncoalveolar (FLBA); determinação da concentração das citocinas IL-4, IL-5, IL-10, IL-13, IL-17A e IFN-y no FLBA por citometria de fluxo (Cytometric Bead Array - CBA); avaliação da expressão de IL-4, IL-5, IL-10, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas e avaliação da atividade proteolítica de tripsina-like, MMP-1 e MMP9. A significância foi considerada p < 0,05. RESULTADOS: O tratamento com rBmTI-A nos animais sensibilizados reduziu a atividade proteolítica de tripsina no tecido pulmonar; a resposta máxima de Rrs e Ers; o número de polimorfonucleares e a concentração de IL-5, IL-10, IL-13 e IL-17A no FLBA; a expressão de IL-5, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; o número de eosinófilos e a fração de fibras colágenas e elásticas nas vias aéreas do grupo OVA+rBmTI-A comparado ao grupo OVA (p < 0,05). CONCLUSÃO: O rBmTI-A atenuou a hiper-responsividade brônquica, a inflamação e o remodelamento nesse modelo experimental de inflamação pulmonar alérgica crônica. Embora mais estudos precisem ser realizados, este inibidor pode contribuir como potencial ferramenta terapêutica para o tratamento de asma / INTRODUCTION: Asthma still affects an increasing number of individuals and can be very serious and sometimes fatal. Despite the improved diagnostic efficiency and therapeutic efficacy, most severe asthmatics do not have complete symptom control with available therapies. Some studies suggest the role of serine protease inhibitors in various inflammatory processes, such as Boophilus microplus trypsin inhibitor (BmTIA). AIMS: To evaluate whether rBmTI-A serine protease inhibitor recombinant modulates bronchial hyperresponsiveness to methacholine, airway inflammation and remodeling in an experimental model of chronic allergic lung inflammation. METHODS: Balb/c mice were divided in four groups: SAL (saline), OVA (sensitized with ovalbumin), SAL+rBmTI-A (control treated with rBmTI-A) and OVA+rBmTI-A (sensitized with ovalbumin and treated with rBmTI-A). On days 0 and 14 of the protocol the animals received intraperitoneal injection (i.p) of saline (0.9% NaCl) (SAL and SAL+rBmTI-A) and ovalbumin (50 ug/mL) (OVA and OVA+rBmTI-A). On days 22, 24, 26 and 28 the groups were submitted to inhalations with saline (0.9% NaCl) or ovalbumin (10 mg/ml) and were treated with a rBmTI-A (35.54 pmol in 50 uL of saline or just saline) by nasal instillation, on the days 22 and 28. On day 29, the following analysis were performed: hyperresponsiveness to methacholine and the maximal resistance and elastance responses of the respiratory system were obtained; quantification of the total number of cells, macrophages, lymphocytes and polymorphonuclear in bronchoalveolar lavage fluid (FLBA); determination of the cytokines IL-4, IL-5, IL-10, IL-13, IL-17A and IFN-y concentration in FLBA by Cytometric Bead Array (CBA); IL4, IL-5, IL-10, IL-13, IL-17, MMP-9 and TIMP-1 expression in the airways; histopathological analysis of the lung for quantification of eosinophils, collagen and elastic fibers and evaluation of trypsin-like, MMP-1 and MMP9 proteolytic activity. Significance was considered when p < 0.05. RESULTS: The treatment with rBmTI-A in sensitized animals reduced: the proteolytic activity of trypsinlike in lung tissue, the maximum response of Rrs and Ers, the number of polymorphonuclear cells and the concentration of IL-5, IL-10, IL-13 and IL-17A in FLBA, the expression of IL-5, IL-13, IL-17, MMP-9 and TIMP-1 in the airways, the number of eosinophils and the fraction of collagen and elastic fibers in the airways of the OVA + rBmTI-A group compared to the OVA group (p < 0.05). CONCLUSION: rBmTI-A attenuated bronchial hyperresponsiveness, inflammation and remodeling in this experimental model of chronic allergic pulmonary inflammation. Although more studies need to be performed, this inhibitor may contribute as a potential therapeutic tool for the asthma treatment

INTRODUÇÃO: A asma ainda acomete um número crescente de indivíduos, podendo ser muito grave e, algumas vezes, fatal. A despeito da melhor eficiência diagnóstica e eficácia terapêutica, a maioria dos asmáticos graves não obtém controle total dos sintomas com as terapias disponíveis. Alguns estudos sugerem a atuação de inibidores de serinoproteases em diversos processos inflamatórios, entre estes inibidores encontra-se o Boophilus microplus trypsin Inhibitor (BmTI-A). OBJETIVO: Avaliar se o recombinante do inibidor de serinoproteases rBmTI-A modula a hiperresponsividade brônquica à metacolina, inflamação e remodelamento das vias aéreas em um modelo experimental de inflamação pulmonar alérgica crônica. MÉTODOS: Camundongos Balb-c foram divididos em 4 grupos: SAL (salina), OVA (sensibilizados com ovoalbumina), SAL+rBmTI-A (controle tratados com rBmTI-A ) e OVA+rBmTI-A (sensibilizados com ovoalbumina e tratados com rBmTI-A). Nos dias 0 e 14 do protocolo, os animais receberam injeção intraperitoneal (i.p) de salina (0,9% NaCl) (SAL e SAL+rBmTI-A) e ovoalbumina (50 ug/mL) (OVA e OVA+rBmTI-A). Nos dias 22, 24, 26 e 28 foram submetidos à inalação com salina (0,9% NaCl) ou ovoalbumina (10 mg/ml) e foram tratados com rBmTI-A (35,54 pmol em 50 uL de NaCl) ou apenas salina, via instilação nasal, nos dias 22 e 28. No dia 29, foram realizadas as seguintes análises: hiper-responsividade à metacolina e respostas máximas de resistência e elastância do sistema respiratório; quantificação do número total de células, macrófagos, linfócitos e polimorfonucleares no fluido do lavado broncoalveolar (FLBA); determinação da concentração das citocinas IL-4, IL-5, IL-10, IL-13, IL-17A e IFN-y no FLBA por citometria de fluxo (Cytometric Bead Array - CBA); avaliação da expressão de IL-4, IL-5, IL-10, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; análise histopatológica do pulmão para quantificação de eosinófilos, fibras colágenas e elásticas e avaliação da atividade proteolítica de tripsina-like, MMP-1 e MMP9. A significância foi considerada p < 0,05. RESULTADOS: O tratamento com rBmTI-A nos animais sensibilizados reduziu a atividade proteolítica de tripsina no tecido pulmonar; a resposta máxima de Rrs e Ers; o número de polimorfonucleares e a concentração de IL-5, IL-10, IL-13 e IL-17A no FLBA; a expressão de IL-5, IL-13, IL-17, MMP-9 e TIMP-1 nas vias aéreas; o número de eosinófilos e a fração de fibras colágenas e elásticas nas vias aéreas do grupo OVA+rBmTI-A comparado ao grupo OVA (p < 0,05). CONCLUSÃO: O rBmTI-A atenuou a hiper-responsividade brônquica, a inflamação e o remodelamento nesse modelo experimental de inflamação pulmonar alérgica crônica. Embora mais estudos precisem ser realizados, este inibidor pode contribuir como potencial ferramenta terapêutica para o tratamento de asma / INTRODUCTION: Asthma still affects an increasing number of individuals and can be very serious and sometimes fatal. Despite the improved diagnostic efficiency and therapeutic efficacy, most severe asthmatics do not have complete symptom control with available therapies. Some studies suggest the role of serine protease inhibitors in various inflammatory processes, such as Boophilus microplus trypsin inhibitor (BmTIA). AIMS: To evaluate whether rBmTI-A serine protease inhibitor recombinant modulates bronchial hyperresponsiveness to methacholine, airway inflammation and remodeling in an experimental model of chronic allergic lung inflammation. METHODS: Balb/c mice were divided in four groups: SAL (saline), OVA (sensitized with ovalbumin), SAL+rBmTI-A (control treated with rBmTI-A) and OVA+rBmTI-A (sensitized with ovalbumin and treated with rBmTI-A). On days 0 and 14 of the protocol the animals received intraperitoneal injection (i.p) of saline (0.9% NaCl) (SAL and SAL+rBmTI-A) and ovalbumin (50 ug/mL) (OVA and OVA+rBmTI-A). On days 22, 24, 26 and 28 the groups were submitted to inhalations with saline (0.9% NaCl) or ovalbumin (10 mg/ml) and were treated with a rBmTI-A (35.54 pmol in 50 uL of saline or just saline) by nasal instillation, on the days 22 and 28. On day 29, the following analysis were performed: hyperresponsiveness to methacholine and the maximal resistance and elastance responses of the respiratory system were obtained; quantification of the total number of cells, macrophages, lymphocytes and polymorphonuclear in bronchoalveolar lavage fluid (FLBA); determination of the cytokines IL-4, IL-5, IL-10, IL-13, IL-17A and IFN-y concentration in FLBA by Cytometric Bead Array (CBA); IL4, IL-5, IL-10, IL-13, IL-17, MMP-9 and TIMP-1 expression in the airways; histopathological analysis of the lung for quantification of eosinophils, collagen and elastic fibers and evaluation of trypsin-like, MMP-1 and MMP9 proteolytic activity. Significance was considered when p < 0.05. RESULTS: The treatment with rBmTI-A in sensitized animals reduced: the proteolytic activity of trypsinlike in lung tissue, the maximum response of Rrs and Ers, the number of polymorphonuclear cells and the concentration of IL-5, IL-10, IL-13 and IL-17A in FLBA, the expression of IL-5, IL-13, IL-17, MMP-9 and TIMP-1 in the airways, the number of eosinophils and the fraction of collagen and elastic fibers in the airways of the OVA + rBmTI-A group compared to the OVA group (p < 0.05). CONCLUSION: rBmTI-A attenuated bronchial hyperresponsiveness, inflammation and remodeling in this experimental model of chronic allergic pulmonary inflammation. Although more studies need to be performed, this inhibitor may contribute as a potential therapeutic tool for the asthma treatment

Orientador: Sergio Marangoni, Luis Alberto Ponce Soto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T15:27:30Z (GMT). No. of bitstreams: 1 VilcaQuispe_Augusto_M.pdf: 840674 bytes, checksum: 0ee3f83d8486d73f628e60ca5c8a83fd (MD5) Previous issue date: 2008 / Resumo: Muitas das toxinas isoladas e estudadas em seus efeitos biológicos precisam ser reavaliadas à luz de metodologias otimizadas em HPLC e confirmadas por espectrometria de massas, devido à possível presença de algum componente não estudado. No caso da serpente Bothrops marajoensis, são valorizados os estudos sobre essa espécie devido aos poucos trabalhos realizados, provavelmente por se tratar de uma espécie restrita e rara encontrada na ilha de Marajó, no Pará, e posteriormente em alguns locais litorâneos do Maranhão, aparentemente endêmica. A reprodutibilidade da atividade biológica, através dos efeitos farmacológicos, só é possível com a utilização de frações quimicamente homogêneas que mantenham a integridade da função biológica. No presente trabalho, foi purificada uma nova serinoprotease TLBm, com atividade trombina "like" em um único passo cromatográfico em um sistema de HPLC de fase reversa, com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. A nova serinoprotease foi caracterizada fisico-quimicamente, revelando uma massa molecular de 33332,5 Da por Espectrometria de Massa (MALDI-Tof). Por outro lado, mostrou uma atividade proteolítica perante o substrato cromogênico DL-BA?NA, assim como foi capaz de evidenciar uma atividade fibrinogenolítica frente ao fibrinogênio bovino e hidrolisar a cadeia alfa (a) e beta (ß), comportando-se como uma trombina "like" tipo A e B. Os estudos da atividade cinética mostraram que a serinoprotease com atividade trombina "like" possui um comportamento michaeliano frente ao substrato DL-BA?NA, registrando as constantes cinéticas de Vmax = 2,3x10-1 nmoles p-NA/Lt/min e o KM = 0,52x10-1M. Os estudos do efeito da temperatura sobre a atividade catalítica revelaram que a serinoprotease TLBm apresenta uma ótima atividade em torno de 38ºC e um pH de 8,0. Para confirmar o caráter trombina "like" da TLBm, esta foi inibida pela ação do fenilmetilsulfonil fluoreto (PMSF) e outros inibidores, através dos quais a atividade foi reduzida em mais de 50% (69,89±1,5 %). A análise de composição de aminoácidos mostrou que a serinoprotease TLBm trata-se de uma proteína de caráter ácido ao apresentar um elevado número de aminoácidos ácidos, assim como uma boa quantidade de aminoácidos hidrofóbicos, o que garante a estabilidade conformacional da proteína. A presença de 12 cisteínas sugere a possível presença de 6 pontes dissulfeto. A seqüência N-terminal da serinoprotease com atividade trombina "like" TLBm mostrou um alto grau de homologia seqüencial (55,9 a 79,4 %). No entanto, existem algumas substituições nos aminoácidos (S)12?(H)12 e (V)15?(L)15, os quais poderiam estar relacionados à diferença na atividade biológica estudada aqui ou em outras atividades, o que faria da TLBm uma serinoprotease particular. Com relação ao estudo das atividades biológicas, a TLBm mostrou-se desprovida de atividade hemorrágica, o que é característico desta família de proteínas e reforça a afirmação de esta ser uma serinoprotease. A TLBm apresentou também baixa capacidade inflamatória (edematogênica) e uma alta concentração na letalidade (DL50) intracerebroventricular (i.c.v), evidenciando que a toxina não contribui significativamente com a letalidade do veneno total. A serinoprotease com atividade trombina "like" TLBm tem a propriedade de induzir a agregação plaquetária em plasma rico em plaquetas (PRP) e esse efeito é inibido na presença do PMSF. / Abstract: Many of the isolated and studied toxins in their biological effects need to be revalued, to the light of methodologies optimized in HPLC and confirmed by Mass Spectrometry, due to the possible presence of some component which has not been studied. In the case of the serpent Bothrops marajoensis, its results are valuable due to the fact that there are few works accomplished probably because the treating of a restricted and rare species found at the island of Marajó, in Pará, and later in some coastal places of Maranhão, which seem endemic. The reproduction of the biological activity, through the pharmacological effects, is only possible with the use of chemically homogeneous fractions so that they maintain the integrity of the biological function. In the present work a new serineprotease was purified with activity thrombin like TLBm in a Chromatography step in a system of HPLC of reverse phase, with a high degree of purity and molecular homogeneity, without loss of the biological activity. A new serineprotease was characterized physical-chemically revealing a molecular mass of 33332,5 Da by a Mass Spectrometry (MALDI-Tof), on the other hand, it did show an proteolitic activity before the substratum chromogenic DL-BA?NA, as well as it was capable to evidence an fibrinogenolitic activity in front of the bovine fibrinogen, and hydrolise the alpha (a) and beta (ß) chain which behaved as a thrombin like A and B types. The studies of the kinetic activity showed that the serineprotease with activity thrombin like, possesses a michaeliano behavior in front of the substratum DL-Ba?NA registering the kinetic constants of Vmax = 2,3x10-1 nmoles ?-NA/Lt/min and the KM = 0,52x10-1M respectively. The studies of the effect of the temperature under the catalytic activity, revealed that the serineprotease TLBm was capable to show a great activity around 38ºC and a pH of 8,0. To confirm the character thrombin like of TLBm, it was inhibited by the action of the fenilmetilsulfonil fluoride (PMSF) and other inhibitors, where the activity was reduced in more of the 50% (69,89 ± 1,5%). The analysis of composition of amino acids showed that the serineprotease TLBm is a character protein acid when presenting a high number of acid amino acids, as well as a good amount of amino acids hydrophobic that guarantees the conformational stability of the protein. The presence of 12 cisteinas suggests the possible presence of 6 dissulfeto bridges. The N-terminal sequence of the serineprotease with activity thrombin like TLBm, showed a high degree of sequential homology (55,9 to 79,4%). However there are some substitutions in the following amino acids: (S)12?(H)12 and (V)15?(L)15 which could be related with the difference in the biological activity studied here or with other activities, which would make TLBm a private serineprotease. The studies of biological activity such an as hemorrhage, reveal that the serineprotease is completely deprived which is known is already of this family of proteins. On the other hand the studies of the inflammatory effect (oedematogenic) reveal that the serineprotease possesses a low inflammatory capacity and finally it displays a high concentration in the lethality (DL50) intracerebroventricular (i.c.v.) showing that the toxin doesn't contribute significantly with the lethality of the total poison. The serineprotease with thrombin like activity has the property of inducing the platelet aggregation in platelets-rich plasma (PRP), and that effect is inhibited in the presences of the PMSF. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientadores: Sergio Marangoni, Luis Alberto Ponce Soto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T22:19:51Z (GMT). No. of bitstreams: 1 VilcaQuispe_Augusto_D.pdf: 3256672 bytes, checksum: 5fbbaf997230230025e7a3945d3d9410 (MD5) Previous issue date: 2013 / Resumo: No presente trabalho duas novas serinoproteases com atividade trombina-simile, nomeadas TLBbz e TLBro isoladas a partir de Bothrops brazili e Bothrops roedingeri respectivamente, foram purificadas em um único passo cromatográfico por HPLC de fase reversa com um alto grau de pureza e homogeneidade molecular, sem perda da atividade biológica. Ambas serinoproteases foram caracterizadas fisico-quimicamente, revelando uma massa molecular relativa de TLBbz = 35,13 KDa e TLBro = 20,24 KDa por SDS-PAGE. Ambas apresentaram atividade proteolítica perante o substrato cromogenico DL-Ba?NA. Os estudos da atividade cinética mostraram que as serinoproteases tiveram um comportamento michaeliano frente ao substrato DL-Ba?NA, apresentando uma Vmax = 1,89 nmoles ?-NA/Lt/min e KM = 0,853 mM para TLBbz; e Vmax = 0,0432 nmoles ?-NA/Lt/min e KM = 0,039 mM para TLBro. Ambas serinoproteases apresentam uma atividade ótima em torno de 38 oC e pH 8,0; sendo inibidas pela ação do fluoreto de fenilmetilsulfonila (PMSF) e outros inibidores, através dos quais a atividade trombina-simile foi reduzida em mais dos 50 % (TLBbz = 86.2 % and TLBro = 42.6 %). Ambas TLBbz e TLBro possuem caráter acido ao apresentar um elevado numero de aminoácidos ácidos, assim como uma boa quantidade em numero de aminoácidos hidrofóbicos, o que garante a estabilidade conformacional da proteína. A presença de 12 cisteinas sugere a possível formação de 6 pontes dissulfeto. TLBbz e TLBro evidenciaram uma atividade fibrinogenolitica frente ao fibrinogenio bovino hidrolisando a cadeia alfa (?) e beta (?), comportando-se como uma trombina-simile tipo AB no caso da TLBro e tipo B, por hidrolizar a cadeia beta (?), no caso da TLBbz. Finalmente, estudos da atividade biológica indicam a propriedade de induzir agregação plaquetaria e esse efeito e inibido pela presença de PMSF / Abstract: In this work, two new serine proteases with thrombin-like activity called TLBbz and TLBro from Bothrops brazili and Bothrops roedingeri respectively were purified in a single chromatographic step by reverse phase HPLC with a high degree of purity and molecular homogeneity without loss of biological activity. Both serine proteases have been characterized physico-chemically, showing a relative molecular mass of TLBbz = 35.13 and TLBro = 20.24 kDa by SDS-PAGE. On the other hand, showed a proteolytic activity towards the chromogenic substrate DL-Ba?NA. The studies of the kinetic activity showed that serine proteases have a michaelian behavior when tested with the substrate DL-Ba?NA, with a Vmax = 1.89 nmoles ?- NA/Lt/min and KM = 0.853 mM for TLBbz, and Vmax = 0.0432 nmoles ?-NA/Lt/min and KM = 0.039 mM for TLBro. Both serine proteases have an optimal activity around 38 °C and pH 8,0; and were inhibited by the action of phenylmethylsulfonyl fluoride (PMSF) and other inhibitors, whereby the thrombin-like activity was reduced in more than 50 % (TLBbz = 86.2 % and TLBro = 42.6 %). Both TLBbz and TLBro have acidic character by presenting a large number of aminoacids acids and a good amount of hydrophobic amino acids, which ensures the conformational stability of the protein. The presence of 12 cysteines suggests the possible formation of six disulfide bridges. TLBbz and TLBro showed a fibrinogenolytic activity against the bovine fibrinogen and hidrolise alpha (?) and beta (?) chain, behaving as a thrombin-like AB types in the case of TLBro and hydrolyzing the beta chain (?) behaving as a thrombin-like B types in the case of TLBbz. Finally, studies of biological activity indicate the ability to induce platelet aggregation and this effect is inhibited by the presence of PMSF / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Ce travail porte sur le développement de sondes peptidiques pour le suivi de la protéolyse par spectroscopie de résonance paramagnétique électronique (RPE) et pour l'imagerie in vivo par résonance magnétique rehaussée de l’effet Overhauser (OMRI). Plus précisément, ce travail étudie pour la première fois une famille d’agents d’imagerie appelée « nitroxyde à déplacement de raies spectrales » spécifique d’activités enzymatiques. L'activité protéolytique, entraînant un décalage de 5 G dans les constantes de couplages hyperfins, permet une quantification individuelle des espèces substrat et produit par RPE et une excitation sélective par OMRI. Trois substrats ont été élaborés, montrant une spécificité enzymatique pour l’élastase du neutrophile (NE) (MeO-Suc-Ala-Ala-Pro-Val-Nitroxyde & Suc-Ala-Ala-Pro-Val-Nitroxyde), et pour la chymotrypsine et la cathepsine G (Suc-Ala-Ala-Pro-Phe-Nitroxyde). Les constantes enzymatiques ont montré de bonnes valeurs avec globalement, Km = 28 ± 25 µM et kcat = 19 ± 3 s-1. Ex vivo, l’utilisation des substrats NE en OMRI a révélé un contraste élevé dans les lavages broncho-alvéolaires de souris sous stimulus inflammatoire. Les rehaussements de signaux IRM sont en corrélation avec la sévérité de l’inflammation. L'irradiation à la fréquence RPE de 5425,6 MHz a permis d'accéder à la bio-distribution des substrats in vivo et pourrait ainsi servir d’outil diagnostic. Les perspectives à moyen terme de ce travail reposent sur le développement de l’OMRI à très faibles champs magnétiques en vue d’une application chez l’homme. / This work focuses on substrate-based probes for proteolysis monitoring by Electron Paramagnetic Resonance spectroscopy (EPR) and for in vivo imaging by Overhauser-enhanced Magnetic Resonance (OMRI). More precisely, this work investigates for the first time a family of MRI agents named “line-shifting nitroxide” specific for proteolytic activities. Proteolytic action results in a shift of 5 G in EPR hyperfine coupling constants allowing individual quantification of substrate and product species by EPR and selective excitation by OMRI. Three substrates were worked out, showing enzymatic specificity for neutrophil elastase (MeO-Suc-Ala-Ala-Pro-Val-Nitroxide & Suc-Ala-Ala-Pro-Val-Nitroxide), and for Chymotrypsin/Cathepsin G (Suc-Ala-Ala-Pro-Phe-Nitroxide). Enzymatic constants were remarkably good with globally Km = 28 ± 25 µM and kcat = 19 ± 3 s-1. Ex vivo, the use of NE substrates in OMRI revealed a high contrast in bronchoalveolar lavages of mice under inflammatory stimulus. MRI signal enhancements correlate with the severity of inflammation. Irradiation at the RPE frequency of 5425.6 MHz provided access to the bio-distribution of substrates in vivo and could thus serve as a diagnostic tool. The medium-term perspectives of this work are based on the development of OMRI with very low magnetic fields for human application

<p>Deep vein thrombosis (DVT) and Pulmonary embolism (PE) are responsible for over 900,000 cases and 100,000 deaths each year in the US. Direct fibrinolytic agents such as plasmin are being investigated for their treatment. However, plasmin administration is not widely studied as low plasmin concentrations are rapidly inactivated by antiplasmin in vivo, whereas high plasmin doses would deplete endogenous antiplasmin and impose bleeding risks. Thus, a plasmin delivery system that can achieve efficient clot lysis while minimizing inactivation by antiplasmin and has reduced bleeding risks is needed. To address this, we propose using reversible inhibitors of plasmin that can sequester plasmin from antiplasmin and release it on the surface of a fibrin clot to achieve clot lysis. The inhibition must be tuned such that it is strong enough to protect plasmin from antiplasmin and weak enough to release plasmin at the clot for lysis. To achieve this, we utilize principles of multivalency to synthesize three classes of inhibitors with varying potencies and mechanisms of inhibition: (i) Multivalent benzamidines (ii) Multivalent tranexamic acids (TXA), and (iii) Hetero-multivalent inhibitors having both benzamidine and TXA. Benzamidine is a competitive inhibitor of plasmin’s active site. TXA, on the other hand, is an FDA-approved weak active site inhibitor that is primarily used to disrupt plasmin(ogen) from binding to fibrin on the clot by inhibiting plasmin’s kringle domains. Multivalent inhibitors were synthesized using amine-reactive chemistry, purified using RP-HPLC and confirmed with Mass Spectrometry. Inhibition assays were performed to assess inhibition potency by determining Ki values (inhibition constants). Lower Ki values indicate stronger inhibition. With multivalent benzamidine derivatives, it was observed that changing valency and linker length substantially impacted inhibition and resulted in Ki values ranging from 2.1 to 1,395 μM. Inhibitors of higher valencies and shorter linker lengths exhibited stronger inhibition. Multivalent TXAs of valencies 1 to 16 were also tested and they exhibited Ki values varying from 2.5 to 21,370 μM indicating up to 8,548-fold improvement in inhibition due to valency. It was found that monovalent TXA, primarily a kringle inhibitor, can be converted into a stronger active site inhibitor by multivalency. With hetero-bivalent TXA-dPEG36-AMB, simultaneous binding of benzamidine to the active site and TXA to the kringle domains was achieved to attain improved inhibition. These results indicate that multivalency can significantly alter the potency of inhibitors and can modulate plasmin inhibition for drug delivery.</p>