La quercétine (QCT) est un composé polyphénolique d'origine végétale connu pour ses activités antioxydantes et ses effets bénéfiques sur la santé. Sa solubilité, sa stabilité, sa biodisponibilité et ses activités biologiques peuvent être améliorées par une acylation sélective de ses groupements hydroxylés. Ce travail vise à étudier la possibilité d'une acétylation enzymatique de la QCT par la lipase B de Candida antarctica (CALB), la lipase la plus exploitée industriellement pour des estérifications régio- et énantiosélectives. Dans une perspective d'ingénierie rationnelle de l'enzyme, une démarche de modélisation moléculaire est mise en oeuvre pour mieux comprendre les interactions qui régissent le positionnement et l'orientation du substrat dans le site actif de la lipase. Dans une première partie expérimentale, l'absence d'activité d'acétylation de la CALB envers la QCT, en présence d'un excès d'acétate de vinyle, a été confirmé. Dans une seconde partie, cette inactivité de la CALB a été expliquée à l'aide de simulations de docking et de dynamique moléculaire. Elle résulte d'une orientation inappropriée du donneur d'acyle liée à la sérine catalytique et d'une proximité insuffisante des hydroxyles de la QCT vis-à-vis des résidus catalytiques. L'éloignement de la QCT de la triade catalytique est due à la rigidité de la molécule, l'étroitesse du site actif ainsi qu'à des interactions hydrophobes et électrostatiques entre le substrat et les résidus de la cavité. En revanche, cette approche de simulation moléculaire prédit un bon positionnement des deux substrats dans le site actif de la lipase de Pseudomonas cepacia (PCL), laquelle est capable d'acétyler la QCT. Dans une troisième partie, l'influence de mutations de deux résidus impliqués dans les liaisons de stabilisation hydrophobe de la QCT dans la CALB a été investiguée par simulation. La substitution d'isoleucines par des valines et des alanines conduit à une augmentation du volume de la poche catalytique et une mobilité accrue de la QCT. Mais ces mutations sont insuffisantes pour permettre un positionnement adéquat de l'acétate et de la QCT par rapport à la triade catalytique. La dernière partie focalise sur les interactions électrostatiques entre la QCT et le site actif de CALB. Les orientations du substrat dans la cavité suite à une méthylation ou une acétylation des groupements hydroxyles de la QCT sont précisées / Quercetin (QCT) is a plant-produced polyphenolic compound well-known for its antioxidant activities and beneficial health effects. Its solubility, stability, bioavailability and biological activities may be improved by a selective acylation of its hydroxyl groups. This work aims at studying the possibility of QCT enzymatic acetylation by Candida antarctica lipase B (CALB), the most industrially exploited lipase for regio- and enantioselective esterifications. In prospect of the rational enzyme design, a molecular modeling approach was implemented to understand the interactions that govern the substrate positioning and orientation in the lipase's active site. In a first experimental part, the absence of CALB acetylation activity towards quercetin in excess of vinyl acetate was confirmed. In a second part, this inactivity of CALB was explained by means of docking and molecular dynamics simulations. This results from an inappropriate positioning of the acyl donor linked to the catalytic serine and from an insufficient proximity of QCT hydroxyls vis-à-vis catalytic residues. The distance of QCT from the catalytic triad is due to its rigidity and to the narrow active site as well as to hydrophobic and electrostatic interactions between the substrate and the cavity residues. On the contrary, this molecular simulation approach predicts an appropriate positioning of both substrates in the active site of Pseudomonas cepacia lipase (PCL), which can perform QCT acetylation. In a third part, the impact of mutations of two residues implicated in the stabilization of QCT by hydrophobic interactions in CALB was investigated through simulations. The substitution of isoleucines by alanines and valines led to an increase in the catalytic pocket volume which intensified the mobility of QCT. However, these mutations are insufficient to allow an appropriate positioning of acetate and QCT in relation to the catalytic triad. The last part of this work focuses on the electrostatic interactions between QCT and CALB's active site. The substrate orientation in the cavity following methylation or acetylation of QCT's hydroxyl groups was clarified

Dans le cadre d’un projet de drug design, l’amélioration de la prédiction de l’affinité représente toujours un défi malgré les nombreux efforts déployés dans ce sens. De plus, les constantes cinétiques d’association et de dissociation sont d'un intérêt majeur pour la découverte de nouveaux médicaments, notamment au stade précoce de l'optimisation des molécules afin de mieux évaluer leurs tolérances et efficacités. De par la récente émergence des études de constantes cinétiques, il existe peu de méthodes de prédiction de ces dernières et aucune approche efficace n'a encore été développée pour estimer correctement ces paramètres cinétiques.En relevant ces deux défis, le premier volet de cette thèse consiste au développement de nouvelles méthodes qui permettent dans un premier temps d’améliorer la prédiction de l’affinité par docking flexible et dans un deuxième temps la prédiction des constantes cinétiques d’association et de dissociations (kon et koff) grâce à des simulations de dynamique moléculaire accélérées.Dans le second volet de cette thèse, nous avons conçu de nouveaux inhibiteurs des LIM kinases, cibles émergentes impliquées dans plusieurs physiopathologies incluant la neurofibromatose et le cancer. Nos composés ont de bonnes affinités et sélectivités in vitro, et d’excellentes activités et tolérances évaluées sur des tests cellulaires. / In a drug design project, improving the prediction of affinity is still an issue despite the considerable efforts made in this direction. In addition, binding kinetic constants are of major interest for the discovery of new drugs, in particular at the early stage of molecules optimization to better evaluate their tolerance and efficacy. Due to the recent emergence of the importance of binding kinetics, methods of kinetic rates prediction remain scarce and no efficient computational approach has still been developed to correctly estimate kinetic parameters.In order to challenge these two problematics, the first part of this thesis consists in the development of new methods that allow, first, to improve the prediction of affinity by a flexible docking and, second, to predict the ligand binding/unbinding pathways and binding kinetic rates (kon and koff) by enhanced molecular dynamics simulations.In the second part of this thesis, we have designed novel inhibitors of LIM kinases, emerging targets involved in several pathophysiologies including neurofibromatosis and cancer. Our compounds have good affinities and selectivities in vitro, and excellent activities and tolerances evaluated on cellular tests.

Uma etapa essencial no ciclo de vida do vírus HIV é a integração do DNA viral no cromossomo hospedeiro. Essa etapa é catalisada pela enzima integrase (IN) de 32-kDa. HIV-1 IN é um importante e validado alvo, e as drogas que inibem seletivamente a enzima, quando utilizadas em combinação com os inibidores da transcriptase reversa (RT) e protease (PR), são consideradas altamente eficazes em suprimir a replicação viral. IN catalisa dois processos enzimáticos designados por 3\' processamento e transferência de DNA. Agentes ativos contra integrase, inibindo a etapa de transferência da vertente já estão em fase clínica. O fármaco Raltegravir® é o primeiro nesta nova classe. Os ensaios clínicos no tratamento em novos pacientes têm uma atividade anti-retroviral potente e bem tolerado. Dada a sua potência, segurança e novo mecanismo de ação, os inibidores da integrase representam um importante avanço terapêutico contra o HIV-1. Na presente tese de doutorado, foram realizados estudos quimiométricos utilizando descritores teóricos e QSAR bi- (2D) e tridimensionais (3D) empregando, respectivamente, as técnicas holograma QSAR (HQSAR) e a análise comparativa dos campos moleculares (CoMFA), visando à geração de modelos preditivos para um conjunto de inibidores da integrase do vírus HIV-1. Modelos de QSAR com boa consistência interna, habilidade preditiva e estabilidade foram obtidos em todos os casos. Os modelos gerados, associados às informações obtidas pelos mapas de contribuição 2D e de contorno 3D, são guias químico-medicinais úteis no planejamento de novos inibidores mais potentes e seletivos da integrase do HIV-1. / An essential step in the HIV life cycle is integration of the viral DNA into the host chromosome. This step is catalyzed by a 32-kDa viral enzyme HIV integrase (IN). HIV-1 IN is an important and validated target, and the drugs that selectively inhibit this enzyme, when used in combination with reverse transcriptase (RT) and protease (PR) inhibitors, are believed to be highly effective in suppressing the viral replication. IN catalyzes two discrete enzymatic processes referred as 3\' processing and DNA strand transfer. Agents active against HIV-1, which target the viral integrase by inhibiting the strand transfer step of integration, have now initialized the clinical trials. The Raltegravir® is the first drug in this new class. Clinical trials in treatment-experienced and in treatment-naive patients have shown that raltegravir-containing regimens have potent antiretroviral activity and are well tolerated. Given their potency, safety and novel mechanism of action, integrase inhibitors represent an important advance in HIV-1 therapy. In the present thesis, Bi- and Tridimensional Quantitative Structure-Activity Relationship (QSAR) studies were performed applying chemometric methods based on theoretical descriptors, Comparative Molecular Field Analysis (CoMFA) and Holograma QSAR (HQSAR) techniques, aiming to generate predictive models for a large set of HIV-1 IN inhibitors. QSAR models presenting good internal consistency, predictive power and stability were obtained in all cases. The final models along with the information resulted by 2D contribution and 3D contour maps should be useful in the design of new inhibitors with increased potency and selective within the chemical diversity of the data.

Les polykétides sont des molécules complexes possédant des rôles thérapeutiques divers (antibiotiques, anticancéreux, immunosuppresseurs, etc..). Ces composés sont synthétisés par les polykétides synthases (PKS) qui présentent donc un intérêt certain. Une stratégie prometteuse et en développement, appelée biologie synthétique, consiste en l’ingénierie protéique des PKS pour produire de nouveaux polykétides. Cependant, un prérequis au succès de cette méthode est la compréhension du fonctionnement des PKS. En effet, les PKS sont des systèmes complexes composés de plusieurs sous-unités. Celles-ci comportent chacune un ou plusieurs modules responsable d’un cycle d’extension du polykétide. Les modules sont composés également de plusieurs domaines assurant chacun un rôle biosynthétique. Il est ainsi nécessaire de comprendre comment s’opère la communication entre domaines au sein d’un module. Pour cela, nous avons étudié le module 5 de la PKS synthétisant la virginiamycine M par une approche combinant la diffusion des rayons X aux petits angles (SAXS), la résonance magnétique nucléaire (RMN) et la modélisation par homologie. Ainsi nous sommes parvenus à caractériser la structure en solution du module 5, mais également à positionner les structures à haute résolution des domaines à l’intérieur de l’enveloppe SAXS. De plus, notre étude de la dynamique du module 5 a montré que les deux domaines acyl carrier protein (ACP) composant le module semblent non-équivalents, et que l’ACP5b doit être doté d’une certaine mobilité afin d’être fonctionnel, ceci dû à la flexibilité du linker reliant les deux ACP. L’interaction entre les sous-unités consécutives est également primordiale pour assurer la fidélité de la synthèse des polykétides. Ces interactions sont assurées, au moins en partie, par des domaines de petite taille appelés « domaine de docking (DD) ». Jusqu’alors, les DD avaient été caractérisés uniquement chez les PKS de type cis-AT. Nous avons caractérisé une nouvelle classe de DD, la première chez les PKS trans-AT. Grâce à une approche pluridisciplinaire, nous avons montré que l’un des DD constitue probablement une protéine intrinsèquement désordonnée (IDP) : son interaction avec le DD partenaire induirait son repliement. Nous avons résolu la structure RMN d’une protéine de fusion entre les deux DD, affichant une nouvelle topologie pour une interaction protéine-protéine. Cette interface de docking a ensuite été replacée dans son contexte modulaire par SAXS. Contrairement aux autres DD qui ne forment qu’une seule interface de docking, ces DD forment deux complexes de docking à l’intersection des deux sous-unités. Nos données SAXS nous avons également permis de proposer un modèle de l’interface créée entre deux sous-unités PKS dans laquelle une chambre réactionnelle est formée, qui pourrait servir à protéger des intermédiaires réactifs de polykétide. Des enzymes post-PKS interviennent suite à la synthèse du polykétide pour maturer ce dernier. Cette étape est d’une importance considérable puisqu’elle confère la structure et l’activité finale du polykétide. Dans ce contexte, nous avons mené une étude structure/fonction de l’enzyme post-PKS catalysant la macrocyclisation de l’antibiotique lankacidine C. Après un phasage par SAD sur la protéine séléniée, nous avons résolu la structure de l’enzyme en complexe avec des analogues de substrats, puis procéder à la conception de mutants pour résoudre la structure de l’enzyme avec son substrat naturel / Polyketides are structurally complex molecules which exhibit diverse therapeutic activities (antibiotic, antitumor, immunosuppressant…). These compounds and the enzymes responsible for their synthesis, the polyketide synthases (PKSs), are thus of significant biomedical interest. An emerging though promising strategy for the generation of novel polyketides is the engineering of the PKS proteins, an approach called synthetic biology. Nevertheless, a fundamental understanding of the mode of operation of the PKS enzymes is directly correlated with the success of this methodology. Indeed, PKSs are molecular-scale assembly lines which are composed of several subunit, each of which includes one or several modules catalyzing a polyketide elongation cycle. The module themselves are composed of several domains each with a specific role in the biosynthesis. It is therefore necessary to understand how the domains within a module communicate with each other. To address this question, we studied module 5 of the PKS responsible for virginiamycin M using an approach combining small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) and homology modeling. This strategy allowed us to characterize the solution structure of module 5, but additionally to position high-resolution domain structures inside the SAXS envelopes. We also studied the dynamics of module 5, demonstrating that the two component acyl carrier proteins (ACPs) appear to be non-equivalent, and that the function of ACP5b is likely dependent on the mobility conferred on it by the flexible linker between the two domains. The interaction between the consecutive subunits is also critical to ensuring the fidelity of polyketide synthesis. This communication is assured, at least in part, by small domains called docking domains (DD), which have to date only been characterized from cis-AT PKS. Here, we have identified a new class of DD, the first shown to be present in trans-AT PKSs. Using a multidisciplinary approach, we have demonstrated that the N-terminal DD (VirFG NDD) is likely to be an intrinsically disordered protein (IDP), whose interaction with its partner DD (VirA CDD) induces its folding. We have solved the NMR structure of a fusion protein between the two DD, revealing a new topology for a protein-protein interaction. This docking interface was then placed into its modular context by SAXS. In contrast to the other classes of DD which form a single docking complex, this new type of DD gives rise to two docking interfaces at the intersubunit junction. Finally, our SAXS data have allowed us to propose a model for the complete interface between two PKS subunits in which a reaction chamber is formed, which may allow reactive polyketide intermediates to be protected. Post-PKS enzymes catalyze maturation of the polyketide after its release from the last module of the PKS. This processing is critical as it yields the final structure and activity of the polyketide. In this context, we conducted a structure/function study of the post-PKS enzyme catalyzing the macrocyclisation of the antibiotic lankacidine C. After phasing by SAD using a seleniated protein, we solved the structure of the enzyme in complex with substrate analogues. We then proceeded to site-directed mutagenesis of specific residues, in order to solve the structure of the enzyme in complex with its natural substrate

Foram determinadas por meio de difração de raios x as estruturas de cinco compostos azaçúcares. Foram estudadas as interações envolvidas na formação das redes cristalinas em cada um dos compostos analisados. Foi encontrado que nos compostos azaçúcares estudados, as interações principais são as ligações de hidrogênio do tipo C-H···O e C-H···π. Este comportamento foi verificado usando ferramentas como as superfícies de Hirshfeld e os gráficos de impressão digital. Realizou-se o estudo de docking molecular dos compostos azaçúcares com respeito à enzima purina nucleosídeo fosforilase (PNP). Foi determinado que estes compostos têm a capacidade de entrar no sitio ativo da PNP. O estudo das interações dos cinco azaçúcares com a PNP mostrou que estes compostos apresentam as mesmas interações presentes em inibidores da PNP já reportados. / Structures of five azasugars were determined by X-ray diffraction. Crystal network interactions were analyzed for each compound. The main interaction found for these azasugar compounds is hydrogen bond as C-H···O e C-H···π. This behavior was verified by tools as Hirshfeld surface and 2D finger print plots. Molecular docking was performed for azasugar compounds in Purine Nucleoside phosphorylase (PNP). This study confirmed that these compounds are available to enter to the PNP active site. Interactions exploration showed the same interactions for the azasugars studied and for already known PNP inhibitors.

Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de α-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

No capítulo 1, a modelagem HQSAR, a docagem e os estudos de ROCS foram construídos utilizando uma série de 57 inibidores de cruzaína. O melhor modelo HQSAR (q2 = 0,70, r2 = 0,95, r2test = 0,62, q2rand. = 0,09 and r2rand. = 0,26) foi utilizado para predizer a potência de 121 compostos extraídos da literatura (conjunto de dados V1), resultando em um valor de r2 satisfatório de 0,65 para essa validação externa. Uma validação externa adicional foi empregada utilizando uma série de 1223 compostos extraído dos bancos de dados ChEMBL e CDD (conjunto de dados V3); nessa validação externa o valor de AUC (área sob a curva) para a curva ROC foi de 0,70. Os mapas de contribuição, obtidos para o melhor modelo HQSAR 3.4, estão de acordo com as predições do modo de interação e com as bioatividades dos compostos estudados. Nos estudos de ROCS, a forma molecular utilizada como filtro, foi útil na rápida identificação de modificações moleculares promissoras para inibidores de cruzaína. O valor de AUC obtido com a curva ROC foi de 0,72, isso indica que o método foi muito eficiente na distinção entre inibidores ativos e inativos da enzima cruzaína. Em seguida, o melhor modelo HQSAR foi utilizado para predizer os valores de pIC50 para novos compostos. Alguns dos compostos identificados, utilizando esse método, demonstraram valores de potência calculada maior do que a série de treinamento em estudo. No capítulo 2, os efeitos sobre a potência na inibição da enzima cruzaína pela substituição de um grupo nitrila como warhead por outros grupos foi avaliada. Com a síntese de 20 compostos do tipo dipeptidil, avaliou-se a relação estrutura-atividade (SAR), baseado na troca do grupo warhead na porção P1\'. O grupo oxima foi mais potente que o grupo correspondente nitrila em 0,7 unidades logarítmicas. Os compostos do tipo dipeptidil aldeídos e azanitrila obtiveram potências mais elevadas do que o correspondente dipeptidil nitrila em duas de magnitude. Os compostos dipeptidil alfa-beta insaturados foram menos potentes do que o correspondente dipeptidil nitrila. No capítulo 3, estratégias de química medicinal foram empregadas nas sínteses de 23 novos análogos, contendo o esqueleto básico de imidazopiridina. Sete e doze compostos sintetizados exibiram EC50 <= 1µM in vitro contra os parasitos Tripanosoma cruzi (T. cruzi) e brucei (T. brucei), respectivamente. Com os resultados promissores de atividade biológica in vitro, citotoxicidade, estabilidade metabólica, ligação proteica e propriedades farmacocinéticas, o composto 41 foi selecionado como candidato para os estudos de eficácia in vivo. Esse composto foi submetido em um modelo agudo da infecção com T. cruzi em ratos (cepa Tulahuen). Depois de estabelecida a infecção, os ratos foram dosados duas vezes ao dia, durante 5 dias; e monitorados por 6 semanas usando um sistema de imagem in vivo IVIS (do inglês, \"In Vivo Imaging System\"). O composto 41 demonstrou inibição parasitária comparável com o grupo de treinamento dosado com benzonidazol. O composto 41 representa um potencial líder para o desenvolvimento de novos fármacos para o tratamento de tripanossomíases. / In chapter 1, the HQSAR, molecular docking and ROCS were applied to a dataset of 57 cruzain inhibitors. The best HQSAR model (q2 = 0.70, r2 = 0.95, r2test = 0.62, q2rand. = 0.09 and r2rand. = 0.26) was then used to predict the potencies of 121 unknown compounds (the V1 database), giving rise to a satisfactory predictive r2 value of 0.65 (external validation). By employing an extra external dataset comprising 1223 compounds (the V3 database) either retrieved from the ChEMBL or CDD databases, an overall ROC AUC (area under the curve) score well over 0.70 was obtained. The contribution maps obtained with the best HQSAR model (model 3.4) are in agreement with the predicted binding mode and with the biological potencies of the studied compounds. We also screened these compounds using the ROCS method, a Gaussian-shape volume filter able to identify quickly the shapes that match a query molecule. The AUC obtained with the ROC curves (ROC AUC) was 0.72, indicating that the method was very efficient in distinguishing between active and inactive cruzain inhibitors. These set of information guided us to propose novel cruzain inhibitors to be synthesized. Then, the best HQSAR model obtained was used to predict the pIC50 values of these new compounds. Some compounds identified using this method has shown calculated potencies higher than those which have originated them. In chapter 2, the effects on potency of cruzain inhibition of replacing a nitrile group with alternative warheads were explored; with the syntheses of 20 dipeptidyl compounds, we explored the structure activity relationships (SAR) based on exchanging of the warhead portion (P1\'). The oxime was 0.7 units more potent than the corresponding nitrile. Dipeptide aldehydes and azadipeptide nitriles were found to be two orders of magnitude more potent than the corresponding dipeptide nitriles. The vinyl esters and amides were less potent than the corresponding nitrile by between one and two orders of magnitude. In chapter 3, we synthesized 23 new imidazopyridine analogues arising from medicinal chemistry optimization at different sites on the molecule. Seven and twelve compounds exhibited an in vitro EC50 <= 1µM against Trypanosoma cruzi (T. cruzi) and Trypanosoma brucei (T. brucei) parasites, respectively. Based on promising results of in vitro activity (EC50 &lt; 100 nM), cytotoxicity, metabolic stability, protein binding and pharmacokinetics (PK) properties, compound 41 was selected as a candidate for in vivo efficacy studies. This compound was screened in an acute mouse model against T.cruzi (Tulahuen strain). After established infection, mice were dosed twice a day for 5 days, and then monitored for 6 weeks using an in vivo imaging system (IVIS). Compound 41 demonstrated parasite inhibition comparable to the benznidazole treatment group. Compound 41 represents a potential lead for the development of drugs to treat trypanosomiasis.

Tropomiosina (Tm) é uma das proteínas que compõe o filamento fino (actina, Tm, Troponina) do sistema muscular esquelético e desempenha um importante papel na regulação da contração muscular. Tm é um coiled-coil de 284 resíduos que forma longos homopolímeros lineares através da sobreposição de onze resíduos entre os terminais de Tms adjacentes (Interação cabeça-cauda) em condições de baixa força iônica. A presença de vários resíduos carregados (D2, K5, K6, K7, D275, H276 e D280) nas extremidades da Tm sugere que contatos intermoleculares eletrostáticos entre estes aminoácidos podem ter um importante papel na estabilidade dos polímeros. Entretanto, a estrutura do complexo cabeça-cauda demonstra que a maioria dos contatos intermoleculares na interface é de natureza hidrofóbica. A fim de analisarmos a contribuição dos grupos carregados para a estabilidade do complexo cabeça-cauda, construímos fragmentos recombinantes correspondentes à metade amino (ASTm1-142 ) e carboxi (Tm143-284(5OHW269)) terminais da proteína contendo mutações pontuais daqueles resíduos para alanina, e adicionalmente H276 para Glu. Medimos a afinidade entre todas as possíveis combinações destes fragmentos na ausência e presença de íons Mg2+, visto que este cátion está sempre presente em condições fisiológicas e é importante para estabilizar a interação entre Tm e actina. Os efeitos das mutações foram analisados por simulações de docking, desnaturações térmicas e ciclos de duplos mutantes. Os resultados demonstram que os aminoácidos K5, K7 e D280 presentes na interface formam contatos intermoleculares essenciais para a estabilidade do complexo. Enquanto, D2, K6, D275 e H276 não participam na formação de contatos intermoleculares, no entanto, contribuem para a estabilidade da interação cabeça- cauda através de suas interações intramoleculares que atuam na estabilidade das hélices individuais. Os aumentos na estabilidade da metade C-terminal da Tm (Tm143-284(5OHW)) induzidos por Mg2+ foram dependentes das mutações neste trecho da proteína sugerindo a presença de um sítio de ligação para este íon na extremidade carboxi terminal da molécula no trecho que forma a interação cabeça- cauda. Construímos um fragmento menor do C-terminal (Tm259-284(W269)) para acompanharmos mudanças no deslocamento químico induzidas pela ligação do íon usando ressonância magnética nuclear. Os resultados obtidos comprovaram nossa hipótese e nos permitiram definir pela primeira vez que a estrutura da Tm tem um ou mais sítios de ligação Mg2+ em uma região próxima ao resíduo H276 que está localizado entre vários resíduos carregados negativamente que participam da interação cabeça-cauda. Por último, estudamos os efeitos de solventes cosmótropicos (TFE e glicerol) nas estabilidades dos fragmentos da Tm, uma vez que a instabilidade (flexibilidade) da extremidade C-terminal é importante para a formação do complexo cabeça-cauda. Observamos que TFE, porém não glicerol, reduziu a afinidade entre os terminais. Ambos os co-solventes induziram aumentos na estabilidade dos fragmentos, no entanto, apenas TFE induziu um aumento no conteúdo de &#945;-hélice e causou uma redução significativa na cooperatividade de desenovelamento das proteínas. Estes resultados indicam que estes compostos orgânicos estabilizam as estruturas dos fragmentos individuais da Tm de maneiras diferentes e que estas diferenças podem estar relacionadas aos diferentes efeitos observados na formação da interação cabeça-cauda. / Tropomyosin (Tm) is a protein component of the skeletal muscle thin filament (actin, Tm, Troponin) which has an important role in the regulation of muscle contraction. Tm is a dimeric coiled-coil (284 aminoacids) which forms long linear homopolymers through the overlap of eleven residues of adjacent Tm termini (Head- to-tail interaction) in low ionic strength conditions. The presence of several charged amino acids (D2, K5, K6, K7, D275, H276 e D280) in Tm extremities suggests that electrostatic contacts among those residues may have an important role in the stability of the polymers. Nevertheless, the solution structure of the head-to-tail complex demonstrated that most of the contacts in the interface are hydrophobic. In order to study the contribution of these charged residues to the stability of the head- to-tail complex, we built recombinant fragments corresponding to the amino (ASTm1-142) and carboxy (Tm143-284(5OHW269)) termini containing single mutations of those amino acids to alanine, and additionally a substitution of H276 for Glu. We measured the binding affinities among all possible combinations of wild-type and mutant fragments in the absence or presence of Mg2+ ions. This cation is always physiologically present in the muscle and it is known to strengthen the binding of Tm to actin. The effects of the mutations were analyzed by protein-protein docking, thermodynamic cycles and thermal denaturations. The results show that residues K5, K7 and D280 are essential to the stability of the complex. Though D2, K6, D275 and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to the complex stability by modulating the stability of the helices at the Tm termini. Mg2+-induced increases in stability of the C- terminal were sensitive to mutations in residues located in the head-to-tail overlap region, suggesting that Mg2+ ions may bind specifically to the carboxy extremity of the protein. We produced a small peptide (Tm259-284(W269)) to follow amide chemical shift perturbations upon Mg2+ binding by nuclear magnetic resonance measurements. The results obtained with this peptide allowed us to define for the first time that the Tm structure has one or more Mg2+ binding sites in a region centered in the vicinity of H276 in which are located several negatively charged residues that participate in the head-to-tail interaction. We also studied the effects of kosmotropic co-solvents (TFE and glycerol) in the stability of Tm fragments, as the instability (flexibility) of the C- terminal region has been pointed as important for the formation of the head-to-tail complex. We observed that TFE, but not glycerol, reduces the affinity between the termini. Both TFE and glycerol increased the stability of the isolated N- and C- terminal fragments; however, only TFE caused an increase in the helical content and a significant reduction in the cooperativity of unfolding of the proteins. Our results show that these two co-solvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their different effects on head-to-tail complex formation.

Bakgrund:                   För att stärka sin position på marknaden önskar Staples Sweden AB utöka sitt artikelsortiment till att omfatta 1 000 000 artiklar. För att möjliggöra sortimentsexpansionen behövs beslutsunderlag rörande hantering och kostnadsstruktur identifieras. Syfte:                          Syftet med studien är att utveckla en modell vid val och utformning av distributionssätt för att föreslå en distributionsslösning. Syftet är också att genom en kostnadsanalys identifiera orderkostnadsstrukturen för den föreslagna distributionslösningen. Metod:                        Vid genomförandet av studien tillämpades en fallstudiedesign. Det empiriska materialet samlades in genom intervjuer utförda på Staples Sweden AB. Slutsats:                      Vid tillämpningen av modellen som utvecklades i studien framkom att distributionslösningen ska utgöras av direktleveranser, konsoliderade direkleveranser, ”cross-docking”, ”cross-docking” med buffert och traditionell lagerhållning. Orderkostnaden skiljde sig mellan distributionssätten där ”cross-docking” var billigast och direktleveransvarianterna var dyrast. / Background:               To strengthen its position on the market, Staples Sweden AB seeks to expand its product range to include one million articles. The problem is that no one in the company knows how such a variety of items should be managed and how the structure of order costs would change. Based on these problems, the task for the study arose. Purpose:                      The thesis aims to develop a model for the selection and design of distribution methods for proposing a solution that can manage one million articles. It also aims to identify the structure of order cost for the proposed solution through a cost analysis. Methodology:             The study was conducted by applying a case study design. The empirical data were collected through interviews conducted at the Staples Sweden AB. Conclusion:                 In the application of the developed model showed that the distribution solution should consist of direct shipping, consolidated direct shipping, cross-docking, cross-docking with buffer and traditional warehousing. Order cost differed between distribution where "cross-docking" showed to be the cheapest and direct shipping variants were the most expensive.

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