abstract: Acute Myeloid Leukemia (AML) is a disease that occurs when genomic changes alter expression of key genes in myeloid blood cells. These changes cause them to resume an undifferentiated state, proliferate, and maintain growth throughout the body. AML is commonly treated with chemotherapy, but recent efforts to reduce therapy toxicity have focused on drugs that specifically target and inhibit protein products of the cancer’s aberrantly expressed genes. This method has proved difficult for some proteins because of structural challenges or mutations that confer resistance to therapy. One potential method of targeted therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes. G-quadruplexes are present in the promoter region of many potential oncogenes and have regulatory roles in their transcription. This study analyzes the therapeutic potential of the compound GQC-05 in AML. This compound was shown in vitro to bind and stabilize the regulatory G-quadruplex in the MYC oncogene, which is commonly misregulated in AML. Through qPCR and western blot analysis, a GQC-05 mediated downregulation of MYC mRNA and protein was observed in AML cell lines with high MYC expression. In addition, GQC-05 is able to reduce cell viability through induction of apoptosis in sensitive AML cell lines. Concurrent treatment of AML cell lines with GQC-05 and the MYC inhibitor (+)JQ1 showed an antagonistic effect, indicating potential competition in the silencing of MYC. However, GQC-05 is not able to reduce MYC expression significantly enough to induce apoptosis in less sensitive AML cell lines. This resistance may be due to the cells’ lack of dependence on other potential GQC-05 targets that may help upregulate MYC or stabilize its protein product. Three such genes identified by RNA-seq analysis of GQC-05 treated cells are NOTCH1, PIM1, and RHOU. These results indicate that the use of small molecules to target the MYC promoter G-quadruplex is a viable potential therapy for AML. They also support a novel mechanism for targeting other potentially key genetic drivers in AML and lay the groundwork for advances in treatment of other cancers driven by G-quadruplex regulated oncogenes. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2017

Doctor of Philosophy / Department of Biochemistry and Molecular Biophysics / Brian V. Geisbrecht / Neutrophils are our most abundant white blood cells and the first leukocytes to infiltrate sites of infection or damaged/healing tissue. Activation of neutrophils results in the mobilization of several types of granules stored within their cytosol, such as the so-called azurophilic granules, which either fuse with the maturing endophagocytic compartment or are released into the extracellular environment. One of the most abundant component of azurophilic granules is a heme-containing enzyme called myeloperoxidase (MPO), which reduces the H₂O₂ produced by the neutrophil’s respiratory burst to generate cytotoxic hypohalous acids, most typically HOCl. While neutrophil granule enzymes are essential for our innate defenses, neutrophil-driven inflammation outside this beneficial context lies at the heart of many non-infectious human diseases. Staphylococcus aureus and closely related species are highly adapted to their hosts and have evolved many strategies to resist opsonization and phagocytosis. S. aureus shows resistance to killing following uptake into the phagosome, which suggests that the bacterium can actively evade specific intracellular killing mechanisms used by neutrophils. Recent work found a highly conserved S. aureus protein, SPIN (for Staphylococcal Peroxidase INhibitor), that specifically binds and inhibits MPO [1]. This study was focused on characterizing the structure/function relationship for MPO inhibitors, SPIN proteins. To identify key residues for SPIN function in more detail, we examined two types of SPIN proteins using structural methods, direct binding assays, and functional assays for MPO activity: deletion mutants and SPIN proteins originating from divergent staphylococcal species. Together, these studies shed light on the molecular features which determine the specificity of SPIN proteins for MPO and suggest potential avenues for using this information toward the design of synthetic MPO inhibitors. In addition to the focus on targeted inhibition of MPO for its therapeutic value in treatment of a number of significant human inflammatory diseases, our investigations contributed in expanding our knowledge on infection spreading. As a first cellular host defense response, the neutrophil interaction with pathogens are of major interest. Characterization of staphylococcal immune evasion proteins is vital for understanding bacterial survival when encountering neutrophils and their bioactive constituents.

Le travail présenté dans ce manuscrit concerne le développement d’inhibiteurs sélectifs de NO Synthases, des enzymes qui catalysent la formation du monoxyde d’azote (NO) à partir de l’arginine. Trois isoformes ont été identifiées, la NOS neuronale (nNOS), la NOS endothéliale (eNOS) et la NOS inductible (iNOS). Une surproduction de NO dans l’organisme, notamment par la iNOS ou la nNOS, est impliquée dans différentes maladies, tel que le choc septique, l’arthrite, des maladies neurodégénératives ou encore certains cancers. L’inhibition des NOSs apparait donc une approche intéressante dans le traitement de ces maladies. Cependant, cette inhibition doit être sélective pour ne pas interférer avec les fonctions physiologiques des autres isoformes.Les composés synthétisés sont constitués d’une partie analogue du substrat de type S-Ethyl-Isothiocitrulline munie d’une extension sensée interagir dans le canal d’accès du substrat où la conservation entre les trois isoformes est plus faible que dans le site de liaison du substrat. Ces deux parties sont reliées par un lien qui peut être de type amide ou hétérocyclique. Ces composés sont obtenus grâce à une stratégie de synthèse en phase solide via un ancrage par la chaîne latérale, qui permet la synthèse rapide de bibliothèques de composés. Notre premier travail a été de tenter d’optimiser deux inhibiteurs sélectionnés de travaux antérieurs, l’un plutôt sélectif de la iNOS et l’autre de la nNOS. Pour cela, nous avons choisi certaines modifications selon une approche aléatoire. Mais nous avons également entamé une approche plus rationnelle en développant un protocole de docking, basé sur des structures de NOSs issues de la Protein Data Bank et destiné à prédire le mode de liaison des inhibiteurs dans le site actif des enzymes. Nous avons également développé de nouvelles séries de composés. En particulier, nous avons mis au point la synthèse sur support d’analogues où le carbone alpha de l’analogue de substrat est remplacé par un azote (composés de type aza). Nous avons aussi tenté la synthèse en solution de nouveaux analogues du substrat séléniés.Enfin, grâce au modèle de docking, un criblage virtuel de molécules issues de deux bases de données dont celle de la Chimiothèque Nationale a été effectué pour découvrir de nouvelles structures avec un potentiel pouvoir inhibiteur. Nous présentons donc dans ce document la conception et la synthèse de nouvelles séries d’inhibiteurs de NOSs, l’élaboration d’un protocole de docking pour apporter une approche plus rationnelle, et enfin l’évaluation des composés sur les trois enzymes recombinantes et, pour certains, sur deux lignées cellulaires productrices de NO. / This work is focused on the development of selective NO Synthases inhibitors, enzymes, which catalyse the production of nitric oxide (NO) from arginine. Three isoforms have been identified, the. neuronal NOS (nNOS), the endothelial NOS (eNOS) and the inducible NOS (iNOS). An overproduction of NO, in particular by iNOS or nNOS, is involved in many diseases such as septic shock, arthritis, neurodegenerative diseases or some cancers. Therefore, NOS inhibition looks very interesting to treat those diseases. However, this inhibition has to be selective of one isoform to not interfere with the physiological functions of the two others.The synthetic compounds are constituted of both an S-Ethyl-Isothiocitrulline as substrate analogue and an extension expected to interact into the substrate access channel, a less conserved region between the three isoforms compared to the substrate binding site. These two parts are joined together by a peptide bond or a heterocyclic link. These molecules have been obtained thanks to a solid-phase strategy through a side chain anchoring approach. This protocol allows the rapid synthesis of compound libraries. In a first work, we attempted to optimize two inhibitors selected from previous studies. One is rather selective of iNOS and the other to nNOS. We chose some modifications in a random fashion. But we also started a more rational approach by developing a docking protocol, based on NOS structures from the Protein Data Bank and designed to predict the binding mode of the inhibitors in the enzyme active sites.We also prepared new series of compounds. In particular, we developed the solid phase synthesis of derivatives where the alpha carbon of the substrate analogue is replaced by a nitrogen (aza-type compounds).. We also attempted the solution synthesis of new substrate analogues containing selenium.Finally, thanks to the docking model, we performed a virtual screening of molecules from two data bases including the Chimiothèque Nationale, to discover new structures with potential inhibitory activity.Therefore, we present in this manuscript the design and synthesis of new series of NOS inhibitors, the development of a docking protocol to provide a more rational approach, and finally the evaluation of compounds on the three recombinant isoforms and, for some, on two cell lines producing NO.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Cognitive impairment is a mental disorder which is associated with neurodegenerative diseases such as Alzheimer's disease (AD), which is the most common form of dementia. Currently there are no consistent evidence who allow support any measure for the prevention of this disease (DAVIGLUS et al., 2010). As for its treatment, there are methods which can provide relative relief of the symptoms, however, only of palliative nature. Thus, this study aimed to evaluate the n-acetylcysteine (NAC), a molecule with neuroprotective properties, in the treatment and prevention of subsequent dementia of AD, using an experimental model of dementia induced by streptozotocin (STZ) in mice. Initially the effect of NAC on the activity of acetylcholinesterase (AChE) activity in vitro mouse brain was evaluated. The ideal dithiobisnitrobenzoate (DTNB) concentration and pH was 0.3 mM and 7.4, respectively. Linearity in enzyme activity was obtained at acetylthiocholine (ATCh) concentrations ranging from 0.025 to 0.450 mM. Sixty sec prior to the addition of ATCh range to avoid interference DTNB NAC interaction was added to the method. NAC interfered with AChE Vmax starting at the concentration of 75 uM without affecting the Km, featuring a non-competitive inhibition. In a second instance, we evaluated the NAC effect on the short-term cognitive impairment in mice. For this, the animals were divided into four groups and were treated with NAC (50 mg/kg /day v.o.) or saline for nine consecutive days, and with STZ (2.5 mg/kg i.c.v.) or aCSF at the first and third days. The results show that NAC treatment: 1) normalized the latency to find the platform in the water maze (MWM) and to get off the platform in passive avoidance (SDPA) that had been altered in animals that received STZ; 2) normalized the AChE and butyrylcholinesterase (BChE) and restored the acetylcholine (ACh) levels in cortical and hippocampal enzymatic activity potentiated by STZ; 3) protected the brain energy metabolism imbalance induced by STZ. Finally, we evaluated the effect of NAC on the long-term cognitive impairment in mice. To this end, animals were treated with NAC (5 mg/kg/day v.o.) or saline for 30 consecutive days, and with STZ (2.5 mg/kg i.c.v.) or aCSF the first and third days. A treatment with physostigmine (PHY; 0.25 mg / kg / day V.O.) was done in parallel as a positive control measure. Totalizing six groups. Both treatments with NAC and PHY: 1) reduced the latency to find the platform in the water maze (MWM) and increased exploratory time on the new object task (NOR) of those animals that received STZ; 2) normalized the cortical and hippocampal AChE enzymatic activity enhanced by STZ; 3) rescued the synaptic plasticity, recovering the synaptophysin (SYN), microtubule-associated protein type 2 (MAP2) and glial fibrillary acidic protein (GFAP) levels reduced by STZ. Thus, the NAC treatment protected from the cognitive impairment induced by STZ in mice, normalizing the cholinergic activity and reestablishing synaptic plasticity. / O déficit cognitivo é uma desordem mental que está associado a doenças neurodegenerativas, como é a doença de Alzheimer (DA) a qual é a forma mais comum de demência. Atualmente não existem evidências consistentes quem permitam apoiar qualquer medida para a prevenção desta doença (DAVIGLUS e col., 2010). Já para o seu tratamento, existem métodos os quais proporcionam alívio relativo dos sintomas, contudo, de natureza paliativa1. Assim, o presente trabalho visou avaliar a n-acetilcisteína (NAC), uma molécula com propriedades neuroprotetora, no tratamento e prevenção da demência consequente da DA, utilizando-se de um modelo experimental de demência induzida pela estreptozotocina (STZ) em camundongos. Inicialmente avaliou-se o efeito da NAC sobre a atividade da acetilcolinesterase (AChE) cerebral de camundongos in vitro. A concentração de ditiobisnitrobenzoato (DTNB) e pH ideais foi de 0,3 mM e 7,4, respectivamente. Obteve-se linearidade na atividade enzimática com concentrações de 0,025 a 0,450 mM de acetiltiocolina (ATCh). Foram adicionados 60 sec de intervalo prévios à adição da ATCh para evitar a interferência da interação DTNB-NAC. A NAC interferiu na Vmax da AChE a partir da concentração de 75 μM sem modificar a Km, caracterizando uma inibição de forma não-competitiva. Em uma segunda instância, avaliou-se o efeito da NAC sobre o déficit cognitivo a curto prazo em camundongos. Para isso, os animais foram divididos em quatro grupos e foram tratados com NAC (50 mg/kg/dia v.o.) ou salina por nove dias consecutivos e com STZ (2.5 mg/kg i.c.v.) ou FCEa no primeiro e terceiro dias. Os resultados mostram que o tratamento com NAC: 1) diminuiu a latência para achar a plataforma no labirinto aquático (MWM) e aumentou a latência para descer da plataforma na esquiva passiva (SDPA) dos animais que apresentaram-se alteradas nos animais que receberam STZ; 2) restaurou a atividade enzimática da AChE e butirilcolinesterase (BChE) e os níveis de acetilcolina (ACh) corticais e hipocampais potencializadas pela STZ; e 3) protegeu do desequilíbrio do metabolismo energético cerebral induzido pela STZ. Finalmente, foi avaliado o efeito da NAC sobre o déficit cognitivo a longo prazo em camundongos. Para tal, os animais foram tratados com NAC (5 mg/kg/dia v.o.) ou salina por 30 dias consecutivos e com STZ (2.5 mg/kg i.c.v.) ou FCEa no primeiro e terceiro dias. Um tratamento com fisostigmina (PHY; 0,25 mg/kg/dia v.o.) foi realizado em paralelo como medida de controle positivo. Totalizando assim seis grupos. Ambos os tratamentos com NAC e PHY: 1) diminuíram a latência para achar a plataforma no labirinto aquático (MWM) e aumentaram o tempo de exploração no novo objeto (NOR) que apresentaram-se alteradas nos animais que receberam STZ; 2) normalizaram a atividade enzimática da AChE cortical e hipocampal potencializada pela STZ; 3) resgataram a plasticidade sináptica, recuperando os níveis de sinaptofisina (SYN), proteína associada aos microtúbulos do tipo 2 (MAP2) e proteína ácida fibrilar glial (GFAP) diminuídas pela STZ. Desta forma, o tratamento com NAC protegeu do déficit cognitivo induzido pela STZ em camundongos, normalizando a atividade colinérgica e reestabelecendo a plasticidade sináptica.

Submitted by Rita de Cassia Siqueira Bruder (bruder.28@hotmail.com) on 2018-03-22T13:20:34Z No. of bitstreams: 1 TESE DE DOUTORADO 2018.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) / Approved for entry into archive by ROSANGELA APARECIDA LOBO null (rosangelalobo@btu.unesp.br) on 2018-03-23T19:14:35Z (GMT) No. of bitstreams: 1 bruder_rcs_dr_bot.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) / Made available in DSpace on 2018-03-23T19:14:35Z (GMT). No. of bitstreams: 1 bruder_rcs_dr_bot.pdf: 1906745 bytes, checksum: 78920ae0ca10dab6eec3900df2039470 (MD5) Previous issue date: 2018-03-02 / RESUMO Bruder R. Redução da Incidência de Citomegalovírus no esquema Sirolimo associado à Tacrolimo em paciente idoso transplantado renal. Tese (Doutorado). Faculdade de Medicina de Botucatu. Universidade Estadual Paulista, 2018. Introdução: O transplante renal é considerado uma opção de terapia renal substitutiva segura para pacientes acima de 60 anos, entretanto, não há consenso sobre o melhor esquema imunossupressor para o paciente transplantado renal idoso. Objetivo: Avaliar a incidência de infecção por citomegalovírus na combinação de tacrolimo e sirolimo em doses reduzidas comparada com a associação tacrolimo e micofenolato. Métodos: Estudo prospectivo randomizado de centro único comparando a combinação de tacrolimo e sirolimo em dose reduzida (grupo sirolimo) contra tacrolimo e micofenolato (grupo micofenolato). Foram incluídos todos os pacientes transplantados renais maiores de 60 anos. Foram avaliadas a incidência de infecção por citomegalovírus (CMV), a sobrevida do paciente e enxerto, taxas de rejeição e função renal em 12 meses de seguimento. Resultados: Foram randomizados 46 pacientes e analisados 44 casos (dois casos excluídos por não terem sido transplantados). As características basais dos grupos foram semelhantes sendo todos os casos transplantados com doador falecido e a maioria induzida com basiliximab. O grupo micofenolato (n=23) e o grupo sirolimo (n=21) apresentaram sobrevida do paciente e enxerto censurado óbito respectivamente de 95,7% e 100% para o micofenolato e 87,3% e 90,5% para o sirolimo sem diferenças estatísticas. A infecção por CMV ocorreu em 60,9% no grupo micofenolato e 16,7% no grupo sirolimo apresentando um risco relativo 3,54 [1,2 – 10,3] vezes maior no grupo micofenolato, p=0,004. A incidência de rejeição e função renal ao longo de 12 meses foi semelhante entre os grupos. A infecção de ferida operatória, retardo de função do enxerto e proteinúria foram semelhantes entre os grupos. O colesterol total e HDL colesterol foram maiores no grupo sirolimo. Conclusão: No grupo de transplantados renais idosos a combinação de tacrolimo e sirolimo em doses reduzidas resultou na redução de 40% da incidência de infecção por CMV. A função renal em 12 meses, taxa de rejeição e sobrevida do enxerto e paciente foram semelhantes. / ABSTRACT Bruder R. Reduced incidence of cytomegalovirus in the Sirolimus associated with tacrolimus in elderly kidney transplant patient. Tese (Doutorado). Faculdade de Medicina de Botucatu. Universidade Estadual Paulista, 2018. Introduction: Renal transplantation is considered safe for patients over 60 years, however, there is no consensus on the best immunosuppressive regimen in elderly. Objective: To evaluate the incidence of cytomegalovirus infection in the combination of tacrolimus and sirolimus in reduced doses compared to the combination tacrolimus and mycophenolate. Methods: A single-center prospective randomized study comparing the combination of tacrolimo and sirolimo in reduced dose (sirolimo group) against tacrolimo and mycophenolate (mycophenolate group). We included all kidney transplant patients over 60 years of age. The incidence of cytomegalovirus (CMV) infection, patient survival and graft, rejection rates and renal function were evaluated at 12 months of follow-up. Results: 46 patients were randomized and we analyzed 44 cases (two cases excluded because they were not transplanted). Baseline characteristics were similar between groups, all patients were transplanted with deceased donor, and the majority were induced with basiliximab. Mycophenolate group (n = 23) and sirolimo group (n = 21) had patient survival, and death censored graft survival respectively 95.7% and 100% for mycophenolate and 87,3% e 90,5% for the sirolimo without statistical differences. The incidence of CMV infection occurred in 60.9% and 16.7% in mycophenolate and sirolimo group respectively. The relative risk of CMV were 3.54 [1, 2 - 10.3] times greater in mycophenolate group, p=0.004. The incidence of acute rejection and kidney function over 12 months was similar between the groups. Infection of the surgical wound, delayed graft function and proteinuria were similar between groups. The total cholesterol and HDL cholesterol were higher in the sirolimo group. Conclusion: In the elderly renal transplant group, the combination of tacrolimus and sirolimus at reduced doses resulted in a 40% reduction in the incidence of CMV infection. Renal function at 12 months, rejection rate and graft and patient survival were similar.

Introdução: A artrite reumatoide é uma doença inflamatória autoimune associada à complicações sistêmicas como fadiga e perda muscular. Perda muscular pode estar relacionada com a ativação do sistema ubiquitina-proteossomo. O objetivo deste trabalho foi avaliar a perda muscular e o evolvimento do proteossomo no modelo de artrite induzida por colágeno (CIA), com ou sem o tratamento de metotrexato ou inibidor de TNF (etanercepte). Métodos: Camundongos DBA1/J machos foram divididos em 4 grupos (n=8 cada): CIA (salina); ETN (etanercepte, 5.5 /) e MTX (metotrexato, 35 /), tratados duas vezes por semana por 6 semanas, e um grupo controle saudável (CO). Tratamentos iniciaram uma semana após a injeção do booster. Escore clínico, edema da pata traseira e peso corporal foram analisados durante o período experimental. Músculo gastrocnêmio (GA) foi pesado após a morte e usado para quantificar a atividade, níveis proteicos e expressão de mRNA das diferentes subunidades do proteossomo através de ensaio fluorogênico, Western blot e rtPCR, respectivamente. Resultados: Tratamentos reduziram o desenvolvimento da doença, observado através do menor escore clínico e edema da pata traseira nos grupos ETN e MTX. ETN apresentou maior peso corporal do que MTX nas semanas 5 e 7. Músculo GA estava aumentado em ETN do que CIA e MTX, um resultado também observado no peso muscular normalizado. As propriedades catalíticas do proteossomo 26S muscular mostraram um aumento na atividade do tipo caspase nos grupos CIA e MTX. Tecidos musculares de animais MTX demonstraram maiores níveis proteicos das subunidades do proteossomo PSMB8 e PSMB9 e maior expressão gênica de Psmb5, Psmb8 e Psmb9. Por outro lado, a expressão de Psmb6 estava diminuída e de Psmb9 estava aumentada em CIA. Conclusões: Apesar de ambos os medicamentos melhorarem o escore da doença, ETN apresentou um afeito anti-artrítico mais forte e foi o único tratamento capaz de prevenir parcialmente a perda muscular. Ao contrário de ETN, CIA e o tratamento com MTX apresentaram perda muscular e atividade e expressão do proteossomo aumentadas. / Background: Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. The aim of this study was to evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods: Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 /) and MTX (methotrexate, 35 /), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR, respectively. Results: Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX groups. ETN presented higher body weight compared to MTX group at weeks 5 and 7. GA weight was heavier in ETN than CIA and MTX, a result also observed in the normalized muscle weight. The catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups. Muscles tissues of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and higher gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions: Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, CIA and MTX treatment did not prevent muscles loss due to increased proteasome expression and activity.

Os inibidores de proteases desempenham nas plantas funções como: defesa contra ataque de predadores de sementes, regulação de enzimas endógenas e fontes de proteínas e aminoácidos. Muitos destes inibidores são utilizados em estudos bioquímicos, bem como no tratamento de patologias humanas como inflamação e câncer. Neste trabalho, um inibidor de serinoprotease, presente na semente de Stryphnodendron barbatinan (barbatimão), foi purificado, caracterizado e denominado SbKI. Sementes de barbatimão maduras foram trituradas, até a obtenção de uma farinha, e esta foi suspensa em PBS, pH 7,4 (1 :5 m/v), sob agitação por 14 horas a 4°C. O extrato foi centrifugado, filtrado e tratado com PVPP, sendo denominado EB, o qual apresentou inibição da coagulação sanguínea e da atividade de algumas serinoproteases. O inibidor SbKI foi purificado utilizando-se três procedimentos cromatográficos: cromatografia de exclusão molecular (Superdex-75, 10/30), troca iônica (Mono-S HR, 5/5), ambas acopladas em um sistema &TA Purifier e fase reversa (C-18, Waters 250 x 4,6mm) acoplada a um sistema HPLC. Em cada etapa de purificação a presença do inibidor foi monitorada pelos testes de atividade inibitória da tripsina e da coagulação, ambos in vitro. SDS-PAGE, sob condições redutoras, mostrou que o inibidor é formado por duas cadeias polipeptídicas (cadeia pesada e leve) unida por ligação dissulfeto. As cadeias foram separadas pela cromatografia de &se reversa após serem reduzidas e alquiladas. Suas seqüências N-terminais foram determinadas pela degradação de Edman, em seqüenciador automatizado, apresentando alta identidade seqüencial com inibidores do tipo Kunitz de outras leguminosas. A determinação da massa/molecular do inibidor e de suas cadeias isoladas, foram determinadas por espectroscopia de massa (LCtESI-MS system) mostrando massas moleculares de 19.570Da7 15530Da e 4040Da, respectivamente. A espectroscopia de dicroísmo circular (CD) revelou que o inibidor é formado predominantemente por elementos beta e estruturas desordenadas. SbKI foi estável a variações de pHs (2-12) e temperaturas extremas e a temperatura de transição foi calculada em 73,3\" C. A determinação das constantes de inibição (KI) foi realizada para as serinoproteases tripsina (KI = 5,5 nM) e calicreína plasmática (KI = 1,l nM). / Proteinase inhibitors perform many beneficia1 roles in plants such as defense against the attack of seed predators, regulation of endogenous enzymes and sources of proteins and amino acids. Many inhibitors are used in biochemistry research, as well as human pathology treatment such as inflammation and cancer. In this work, a serino proteinase inhibitor found in Stryphnodendron barbatiman seeds (barbatimão) was purified, characterized and denoted SbKI. Mature barbatimão seeds were ground and suspended in PBS pH 7.4 (15 wlv) and stirred for 14 hours at 4OC. The suspension was centrifuged, filtered and treated with PVPP and denoted EB. This EB inhibited blood coagulation and some serine proteinases activities. The inhibitor SbKI was purified by three chromatography step: molecular exclusion (on Supredex-75, 10/30), ion exchange (on Mono-S, 5/5), both connected to AKTA Purifíer System and reversed phase (on C-18, Waters 250 x 4.6 mm) connected to HPLC System. In each purification step the presence of inhibitor was monitored, in vitro, by trypsin and coagulation inhibitory activity. SDS-PAGE, reduced conditions, showed two polypeptide chains (heavy and light chains) linked by one disulphide bridge. The chains were separated by reversed phase chromatography aíter reduced and alquilated. The N-terminal sequence were performed on automated protein sequencer by Edman degradation and showed homology with Kunitz type inhibitors from Leguminosae. Molecular weight of inhibitor and its chains were determined by mass spectrometry (LC/ESI-MS System) and showed molecular weight of 19.570Da, 15.530Da and 4040Da, respectively. Circular dichroism spectroscopy showed SbKI is constituted predominantly by P elements and unordered structures. SbKI was stable over extreme ranges of pH (2-12) and temperature and the transition temperature 73.3\"C investigated by CD and fluorescence emission spectroscopies. Inhibition constants (Ki) were determined by typsin (Ki = 5.5 nM) and human plasmatic kallikrein (Ki = 1.1 mM)

A melhor compreensão dos mecanismos fisiopatológicos e farmacológicos aliados a métodos modernos de investigação tornaram possível a descoberta e o desenvolvimento de fármacos para diversas doenças e disfunções orgânicas em humanos. Os fármacos desenvolvidos atualmente são resultados de intensos esforços em pesquisa por equipes multidisciplinares, impactando diretamente na qualidade de vida das diversas populações no mundo. Nesse cenário, os grupos de pesquisas estabelecidos em Universidades com foco no planejamento de fármacos para doenças tropicais têm crescido. A Malária e a Doença de Chagas figuram com especial importância, a primeira pela expressiva mortalidade mundial, enquanto a segunda pela morbidade e seus impactos na população brasileira. O tratamento de ambas possui limitações que se agravam, seja pelo baixo número de opções terapêuticas, ou pelo desenvolvimento de cepas resistentes. As enzimas investigadas nesse doutoramento, enolase (PfEnolase) de Plasmodium falciparum e gliceraldeído3fosfato desidrogenase de Trypanosoma cruzi (TcGAPDH), são componentes da via glicolítica destes parasitas e são considerados alvos moleculares atrativos para o desenvolvimento de inibidores enzimáticos, dada a importância destas enzimas no processo de obtenção de energia do parasita. Os estudos fundamentamse na busca por modulação seletiva da atividade biológica dos alvos selecionados através do desenvolvimento de novas moléculas bioativas. O estabelecimento de protocolo de expressão e purificação para enzima Pfenolase permitiu sua obtenção em quantidade e pureza suficiente para condução de estudos cinéticos e de triagem biológica, com a identificação de cinco novas classes químicas bastante promissoras; além de ensaios de cristalização, que culminaram na determinação da enzima em diversos complexos cristalográficos. Os dados estruturais produzidos foram fundamentais para condução da abordagem computacional de triagem virtual, que permitiu a identificação de 31 moléculas candidatas a inibidoras de Pfenolase. Avanços significativos foram obtidos também com a enzima TcGAPDH, destacando-se as adaptações nos processos de obtenção da proteína recombinante e ensaio cinético, condução de ensaio de bioprospecção orientada com a identificação e caracterização da molécula isolada (tilirosídeo). Novas condições de cristalização foram identificadas e poderão ser empregadas no processo de obtenção de complexos cristalográficos futuros. Adicionalmente, desenvolveu-se uma ferramenta computacional, Kinecteasy, para processamento automatizado dos dados produzidos das etapas de triagem biológica. Os trabalhos integrados de biologia estrutural e química medicinal desenvolvidos contribuem significativamente para o avanço no processo de planejamento de novos inibidores para as enzimas selecionadas. / A better understanding of the pathophysiological and pharmacological mechanisms together with the modern research methods made possible the discovery and development of drugs for several humans´ diseases. The drugs currently developed are the result of intense efforts in research of multidisciplinary teams having as a direct consequence a remarkable impact on life quality of populations all over the world. In this scenario, research groups established at universities, with their focus on drug development for tropical diseases, are increasing. Malaria and Chagas disease deserve special attention, the former by the expressive world mortality, while the second by the morbidity and its impact on Brazilian population. Treatment for both has limitations, whether by the low number of therapeutic options, or by development of resistance. The target enzymes for this PhD project, enolase (PfEnolase) of Plasmodium falciparum and glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi (TcGAPDH), are essential components of glycolytic pathway and therefore related to the parasite energy production, thus, are considered attractive molecular targets for enzyme inhibitors development. Essentially, the proposed studies seek selective modulation of the target´s biological activity through the development of new bioactive molecules. The expression and purification protocols developed for Pfenolase have allowed us to obtain recombinant protein at suitable yield and purity for conducting screening assays, which has revealed five new chemical classes as Pfenolase inhibitors. Crystallization experiments were successfully conducted and 3D structure were determined for different complexes. Structural data was essential for performing the computational approach of virtual screening, which has allowed us to identify 31 inhibitor candidates for Pfenolase. Significant advances were obtained with TcGAPDH, highlighting the adaptations on recombinant protein protocol and kinetic assay. Assay-guided bioprospecting experiments were successfully performed with identification and characterization of isolated inhibitor (tiliroside). New crystallization conditions were identified and will be employed in future co-crystallization and soaking studies. Additionally, Kinecteasy, a computational tool, were developed for automated data processing of biological screening assays. The structure and medicinal chemistry studies presented here contribute significantly in the process of drug development for the selected enzymes.

A resistência natural do gambá Didelphis marsupialis ao veneno de serpentes se deve a fatores antibiotrópicos presentes em seu soro, do qual DM43 é um dos responsáveis pela inibição da atividade hemorrágica. Com o objetivo de entender melhor o mecanismo de ação desta proteína contra a metaloprotease contida no veneno pretendeu-se, neste trabalho, otimizar as condições de cristalização de DM43, na tentativa de determinar sua estrutura por difração de raios-X. Ensaios de cristalização revelaram que o sulfato de amônio é o precipitante mais eficiente na produção de cristais de DM43. A visualização e indexação dos padrões de difração destes cristais permitiram apenas a determinação dos parâmetros de sua cela unitária, devido a baixa qualidade dos mesmos. Deste modo, os seguintes parâmetros de cela unitária foram encontrados: a = b = 117.34 (0.03) Å, c = 193.39 (0.02)Å, ?=?=90° e ? = 120°, correspondentes ao sistema cristalino trigonal ou hexagonal. A recente determinação da sequência de aminoácidos de DM43, possibilitou modelar sua estrutura tridimensional por homologia utilizando o método de satisfação das restrições espaciais. Os três domínios tipo imunoglobulina de DM43 foram modelados em duas partes a partir da estrutura de referência KIR2DL1(1nkr) homóloga a dois domínios C-terminais de DM43, com apenas 27.72% de identidade. O domínio N-terminal denominado D0, foi modelado separadamente contra o domínio C-terminal de KIR2DL1 com um grau de identidade igual a 28.89%. A partir do programa GRASP e PATCHES, foram detectadas as prováveis regiões de ligação entre as duas partes da molécula e uma possível região responsável pela dimerização no domínio D2 de DM43. Interessantemente, resíduos polares em KIR2DLs que foram substituídos por hidrofóbicos em DM43, concentrando-se em uma face do domínio D2 ausente de sítios de glicosilação, o que coincide com as observações de dimerização do receptor do hormônio de crescimento. Um motivo estrutural característico de receptores hematopoiéticos identificados como WSXWS box, foi encontrado em todos os domínios de DM43, especialmente no domínio D2. Supõe-se que a sua existência esteja relacionada com a orientação relativa dos domínios por manter o primeiro triptofano do motivo posicionado exatamente na interface entre os domínios. Surge ainda uma fita extra (F´), no domínio D2, não pertencente ao enovelamento imunoglobulina devido a presença da Pro273, muito bem conservada, a três resíduos do motivo WSXWS. Esta fita parece desempenhar um papel importante na orientação do motivo pois além de formar ligações de hidrogênio com a fita G do domínio anterior, posiciona corretamente o primeiro triptofano do motivo na interface. Somando-se a isto, a presença de resíduos hidrofóbicos na interface dos domínios D1 e D2pode estar contribuindo para a orientação angular relativa menor que 90° entre os dois domínios. Uma interpretação análoga a de hormônios de crescimento sugere que, os loops entre as fitas AB, CC´ e EF do domínio 1, BC e FG do domínio 2 e o linker entre estes dois domínios, estejam envolvidos na interação da metaloprotease com DM43. / The natural resistance of the opossum, Didelphis marsupialis, towards snake venom is due to antibothropic factors present in the blood serum, of which DM43 is one. It is responsible for the inhibition of the hemorrhagic activity of the venom. With a view to better understanding the mechanism of action of this protein against venom metalloproteases, one of the aims of the present work was to optimize the crystallization conditions of DM43 in order to determine its three-dimensional structure by X-ray diffraction. Crystallization trials revealed that ammonium sulphate was the best precipitant. Visualization and indexing of the diffraction patterns from such crystals only allowed for the determination of the unit cell parameters due to their inherently low quality. The values obtained were a = b = 117.34 (0.03) \'ANGSTRON\', c = 193.39 (0.02) Å, ?=?= 90° e ? = 120°, corresponding to the trigonal or hexagonal crystal systems. The recent determination of the amino acid sequence of DM43 permitted the homology modelling of its three-dimensional structure by satisfaction of spatial restraints. The three immunoglobulin-like domains of DM43 were modelled in two separate parts from the reference structure KIR2DL1 (1nkr), homologous to the two C-terminal domains of DM43, with which its shares 27.72% sequence identity. The N-terminal domain, denominated D0, was separately modelled based on the C-terminal domain of KIR2DL1, with which it shares 28.89% identity. The programs PATCHES and GRASP were used to detect the probable interface region between the two separate parts of the molecule as well as a region probably responsible for dimerisation. Polar residues in KIR2DL1 which had been substituted by hydrophobic residues in DM43 are observed concentrated on one face of the molecule devoid of glycosilation sites (D2) coinciding with the dimerisation interface observed in the growth hormone receptor. A structural motif characteristic of the haematopoetic receptor family, identified as the WSXWS box, was observed to some extent in all three domains of DM43 and most evident in domain D2. It is speculated that the existence of this motif is related to the relative orientation of the domains, by maintaining the first tryptophan of the motif positioned at the domain interface. An additional ?-strand (F) in D2, which is not characteristic of the immunoglobulin fold, is observed due to the presence of the conserved Pro273, three-residues prior to the WSXWS box. This strand appears to play an important role in correctly positioning the motif as it forms hydrogen bonds with strand G of the previous domain thus orientating the first tryptophan of the motif at the interface. The presence of hydrophobic residues at the interdomain interface, may also contribute to stabilizing the acute angular relationship between D1 and D2. An analogous interpretation to that given for the growth hormone receptor, suggests that the loops between ?-strands AB, CC\' and EF of domain D1 and between strands BC and FG do domain D2 plus the linker region, are involved in the binding of metalloproteinase by DM43.

Mesmo com o grande número de estudos relacionados à proteases do subtipo B e de como suas mutações podem interferir na estrutura, na resistência a inibidores e na eficiência catalítica da enzima, existe ainda uma lacuna de como as mudanças polimórficas de proteases de HIV de outros subtipos de HIV-1 interferem nesses fatores. Nesse contexto insere-se esse trabalho, que utilizou proteases de HIV-1 isoladas de pacientes brasileiros HIV-1 infectados com o subtipo F, e outros dois mutantes, sendo que um do subtipo F e outro do subtipo B para ensaios frente a seis inibidores comercialmente disponíveis: amprenavir, indinavir, lopinavir, nelfinavir, ritonavir e saquinavir. Nossos resultados experimentais revelam que os seis inibidores comerciais estudados são significantemente menos ativos para o subtipo F e para as mutantes quando comparados ao subtipo B. Além disso, os valores de vitalidade dessas proteases também são considerados maiores que os obtidos para a proteína selvagem do subtipo B. O acúmulo de mutações comumente detectadas e o polimorfismo natural tornam a protease selvagem do subtipo F cataliticamente suficiente para manter a viabilidade do vírus e garantir alto grau de resistência cruzada frente a todos os inibidores estudados. / Despite years of intense research around the world, HIV continues to represent considerable therapeutical challenge. In order to gain more insights into resistance of polymorphic mutations of existing HIV subtypes toward commercially available pharmaceutics, we studied inhibition of subtypes B and F HIV proteases (PRs) [native and two mutant enzymes clinically identified in Brazilian patients] by six commercial inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir). Our results show that all these inhibitors have significantly higher Ki values for the subtype F HIV PR (Fwt) and both mutant enzymes than that for the B subtype HIV PR (Bwt). Furthermore, the biochemical fitnesses of these proteases, or their vitalities, are also considerably higher than that of Bwt. The accumulation of commonly detected resistant mutations in HIV PRs with natural polymorphisms turns Fwt sufficiently catalytically active to guarantee the virus viability and confers it a large degree of cross resistance against all studied inhibitors.