Human Immunodeficiency Virus (HIV) is the causative agent of the pandemic disease Acquired Immune Deficiency Syndrome (AIDS). HIV acts to disrupt the immune system which makes the body susceptible to opportunistic infections. Untreated, AIDS is generally fatal. Twenty years of research by countless scientists around the world has led to the discovery and exploitation of several targets in the replication cycle of HIV. Many lives have been saved, prolonged and improved as a result of this massive effort. One particularly successful target has been the inhibition of HIV protease. In combination with the inhibition of HIV reverse transcriptase, protease inhibitors have helped to reduce viral loads and partially restore the immune system. Unfortunately, viral mutations leading to drug resistance and harmful side-effects of the current medicines have identified the need for new drugs to combat HIV. This study presents computational efforts to understand the interaction of inhibitors to HIV protease. The first part of this study has used molecular modelling and Comparative Molecular Field Analysis (CoMFA) to help explain the structure-active relationship of a novel series of protease inhibitors. The inhibitors are sulfamide derivatives structurally similar to the cyclic urea candidate drug mozenavir (DMP-450). The central ring of the sulfamides twists to adopt a nonsymmetrical binding mode distinct from that of the cyclic ureas. The energetics of this twist has been studied with ab initio calculations to develop improved empirical force field parameters for use in molecular modelling. The second part of this study has focused on an analysis of the association and dissociation kinetics of a broad collection of HIV protease inhibitors. Quantitative models have been derived using CoMFA which relate the dissociation rate back to the chemical structures. Efforts have also been made to improve the models by systematically varying the parameters used to generate them.

This thesis presents the basic concepts of electronic structure theory and the chemical properties of mercury. The theoretical foundation of DFT and the consequences of relativity are also introduced. The electronic structure of Hg(II) ions, [Hg(L)n(H2O)m]q (L = HO-, Cl-, HS-, S2-) has been studied. We show, in this thesis, that the charge transfer (that is calculated from the hard-soft-acid-base principle (Pearson’s principle)), the total NBO charge and the interaction energies are strongly correlated. Our studies indicate the effect of the solvent on the global electrophilicity, the charge transfer and consequently the interaction strength between Hg(II) and ligand L. The formation constants, logK, of Hg2+−complexes are calculated. The procedure that we follow in this thesis to calculate the formation constants, logK’s, are in good agreement with the extrapolated experimental values. We introduce and explain why it is important adding water molecules explicitly during the calculations of the logK. The recommended logK value of HgS is 27.2. We examined two different types of organic compounds as sensors for heavy metal ions: lumazine (Lm) and 6-thienyllumazine (TLm). We found that the simple calculation of pKa values using DFT methods and implicit solvent models failed to reproduce the experimental values. However, calculated orbital energies and gas phase acidities both indicate that the compound TLm is inherently more acidic than the parent species Lm. We demonstrate that: (1) we need to take in our consideration the population of each tautomer and conformer during the calculations of the pKa values, and (2) thienyl group has indirect effect on the acidity of the proton on N1 in the uracil ring. Last but not least, the fluorescence spectrum of the sensors (L) and their [(L)nM(H2O)m]2+ complexes (L = Lumazine (Lm) and 6-thienyllumazine (TLm) and M = Cd2+and Hg2+) are calculated using time dependent DFT (TDDFT). The results show that TDDFT is in good agreement with experimental results. This chapter provides a new concept in the design of fluorescence turn-on/off sensors that has wider applicability for other systems. Finally, we provide a summary of the works compiled in this thesis and an outlook on potential future work. / October 2015

Mit Aminotransferasen können chirale Amine auf biotechnologischem Weg hergestellt werden. Diese besitzen große Bedeutung als Bausteine für weitere Synthesen in der pharmazeutischen und agrochemischen Industrie. Da natürlich vorkommende Enzyme oft nicht die gewünschte Substratspezifität für bestimmte industrielle Anwendungen besitzen, ist eine Optimierung durch Mutagenese notwendig. Solche Entwicklungen sind jedoch oft mit hohem Zeit- und Kostenaufwand verbunden. Die Optimierung kann entweder ungezielt durch empirische Methoden oder gezielt unter Einbeziehung von Informationen über das Enzym erfolgen. Die notwendigen Daten können als Vorbereitung zu konkreten Produktentwicklungen durch Untersuchungen an potentiell geeigneten Enzymen gewonnen werden. Um einen solchen rationalen Ansatz bei der einer speziellen Amin-Pyruvat-Aminotransferase zu ermöglichen, war es Ziel der vorliegenden Arbeit die Grundlagen für die Veränderung der Substratspezifität dieses Enzyms zu erarbeiten. Zunächst wurden strukturelle Informationen durch ein Homologie-Modell gewonnen und später durch eine experimentell bestimmte Struktur ergänzt. Mit dieser Struktur wurden die Substrat-bindenden Reste identifiziert und zunächst der Einfluss auf die Substratbindung durch ortsgerichtete Mutagenese überprüft. Es konnte gezeigt werden, dass alle acht ausgewählten Aminosäurereste an der Substratbindung beteiligt sind. Zudem wurde unter diesen Positionen nach Mutanten gesucht, die neue Substrate umsetzen können. Eine Reihe von Mutanten wurde identifiziert, die verschiedene neue Substrate umsetzen. Für zwei Positionen konnten eine Reihe von Mutanten identifiziert werden, die neue Substrate akzeptieren. Durch die Art der Seitenketten, die Position der Aminosäuren und der chemischen Struktur der akzeptierten Substrate konnten eine Reihe von Aussagen über den Mechanismus der Substratbindung für diese Amin-Pyruvat-Aminotransferase gemacht werden. Außerdem wurde die Zweckmäßigkeit der eingesetzten theoretischen und experimentellen Methoden für die Anwendung bei Entwicklungen mit Enzymen dieser Klasse gezeigt.

A captação de maltose em bactérias é feita por um sistema transportador do tipo ABC composto por uma proteína ligadora de substrato (MalE), duas proteínas transmembrana e uma ATPase. No presente trabalho descrevemos a clonagem, expressão e análise bioquímica e estrutural da proteína MalE da bactéria fitopatogênica Xanthomonas axonopodis pv citri (Xac) O gene malE de Xac foi clonado em vetor de expressão pET28a, a proteína recombinante foi expressa em Escherichia coli e purificada por cromatografia de afinidade ao níquel. Amostras da proteína solúvel foram analisadas quanto à estrutura secundária, interação com possíveis ligantes, estabilidade frente a diferentes condições físico-químicas. Ensaios de cristalização possibilitaram a obtenção de cristais em diferentes condições, um deles apresentou grupo espacial P6122, mas não foi possível resolver a estrutura. Com base nas estruturas conhecidas de ortólogos de MalE, geramos um modelo estrutural para a proteína de Xac e foram feitas análises quanto à interação com trealose e maltose. Modelos estruturais dos componentes transmembrana (LacF e LacG) e ATPase (UgpC) do sistema transportador de maltose de Xac também foram gerados. Os resultados representam uma contribuição importante para o conhecimento sobre a fisiologia e sistemas de transporte de Xac. / Maltose uptake in bacteria is mediated by an ABC transporter comprising a substrate binding protein (MalE), two transmembrane proteins, and one ATPase. In the present study, we describe the cloning, expression and biochemical as well as structural analyses of the MalE protein of the phytopagen Xanthomonas axonopodis pv citri (Xac). The malE gene of Xac was cloned in the pET28a expression vector, the recombinant protein was expressed in Escherichia coli and, subsequently, purified by nickel affinity chromatography. Samples of soluble protein were analyzed regarding secondary structure, interaction with putative ligants and stability under different physico-chemical conditions. Crystallization trials were carried out under different conditions, one particular condition yielded crystal with a P6122space group, but the structure was not solved. Based on known ortholog structures, a structural model for Xac MalE was obtained allowing interaction with modeled threhalose and maltose. Structural models the transmembrane (LacF and LacG) and ATPase (UgpC) components were also obtained. The present results represent an important contribution to the knowledge of the physiology and transporter systems found in Xac.

Orientador: Kleber Gomes Franchini / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T03:49:49Z (GMT). No. of bitstreams: 1 KosckyPaier_CarlosRoberto_D.pdf: 7798282 bytes, checksum: 043e551d51c3a8309982d9ff59835b10 (MD5) Previous issue date: 2014 / Resumo: A via de sinalização da Calcineurina (Cn), uma fosfatase dependente de cálcio, desempenha papel chave no desenvolvimento, na hipertrofia e no remodelamento patológico do coração. A Calcineurina é negativamente regulada pelas Calsarcinas (CS), uma família de proteínas específicas do músculo estriado, que interagem diretamente com Cn. No entanto, os mecanismos moleculares de inibição de Cn por CS permanecem obscuros. Compreender a estrutura do complexo Cn:CS é fundamental para desvendar esse mecanismo de regulação. Neste trabalho foram combinados ensaios bioquímicos, crosslinking químico acoplado à Espectrometria de Massas (experimentos de MS / MS), análise mutacional e uma estratégia de modelagem computacional para a caracterização estrutural do complexo CnA:CS1 (isto é, constituído pela subunidade A de Cn e a isoforma 1 de CS, ambas murinas). O complexo recombinante foi submetido a crosslinking químico, tripsinizado e analisado por LC-MS/MS. Os dados obtidos foram utilizados em um docking in silico dos modelos de ambos os polipeptídeos, gerando várias poses para o complexo. As poses de menor energia de ligação foram agrupadas de acordo com semelhança estrutural e submetidas à simulação de dinâmica molecular. A superfície de interação identificada em CnA abrangeu as ?-hélices 1, 3 e loops vizinhos, enquanto a superfície correspondente de CS1 compreendeu os loops carboxiterminais das regiões Leu179-Phe185, Phe195-Ser199 e Thr250-Leu264. Notavelmente, a superfície de interação de CnA situa-se muito próxima à folha-? 14, o principal sítio de ligação do motivo PxIxIT do fator de transcrição NFAT, importante efetor da Calcineurina. Experimentos realizados com vários mutantes de CnA (FLAG- CnA) e CS1 (myc -CS1 ) foram utilizados para validar o modelo estrutural do complexo CnA:CS1. Os resíduos Lys40 (CnA) e Glu254 (CS1) foram identificados como críticos para a estabilidade do complexo. O modelo gerado neste estudo apoia a hipótese de que CS1 interage com um sítio alostérico para inibir a atividade de CnA / Abstract: Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical crosslinking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd ?-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the ?-sheet 14, the main binding site for the PVIVIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-β1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-β1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-β1 to its TGF-β receptor-I (TβR-I) and -II (TβR-II). However, sequential addition of sHA3 to the TβR-II/TGF-β1 complex led to a significantly stronger recruitment of TβR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TβR-I when added sequentially. For the first time bioactivity of TGF-β1 in conjunction with sHA was investigated at the receptor level. TβR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-β1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-β1-driven skin fibrosis.

Orientadores: Susanne Rath, Rogério Custódio / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T17:08:24Z (GMT). No. of bitstreams: 1 Barros_LeonardoAugustode_D.pdf: 4314419 bytes, checksum: 93a1b964e0a2949ef6a2379990e2f089 (MD5) Previous issue date: 2014 / Resumo: Esse trabalho teve como objetivo principal otimizar e sintetizar polímeros de impressão molecular (MIP) para serem empregados em processos de extração em fase sólida (SPE), visando a determinação de diuréticos tiazídicos em urina. Cálculos teóricos de modelagem molecular, usando o programa computacional Gaussian 09 e os métodos DFT e PCM, no nível B3LYP e conjunto de base 6-31G(d), foram realizados para selecionar o monômero funcional (MF) e o solvente porogênico mais adequados para serem utilizados na síntese dos MIP. Para o desenvolvimento dos MIP compatíveis com água, foi utilizado hidroclorotiazida, clorotiazida ou hidroflumetiazida como molde, acrilamida como MF, etilenoglicol dimetacrilato como reagente de ligação cruzada (RLC) e tetraidrofurano como solvente porogênico. Foram avaliados alguns parâmetros que afetam a eficiência do polímero de impressão, tais como a quantidade de MF e a natureza do RLC. Foram construídas as isotermas de adsorção para cada um dos polímeros sintetizados e foi avaliada a seletividade dos MIP frente a análogos estruturais dos moldes. Os polímeros foram caracterizados por infravermelho com transformada de Fourier, 13C RMN, microscopia eletrônica de varredura, porosimetria de sorção de nitrogênio e análise termogravimétrica. Os MIP foram empregados como fase estacionária em SPE para a determinação de diuréticos tiazídicos em urina por cromatografia líquida de alta eficiência e os mesmos apresentaram seletividade cruzada em relação aos análogos estruturais / Abstract: This work aimed to optimize and synthesize molecularly imprinted polymers (MIP) to be employed in processes of solid phase extraction (SPE), for the determination of thiazide diuretics in urine. Theoretical calculations of molecular modeling, using the Gaussian 09 software and the density functional theory and PCM methods, at the B3LYP/6-31G(d) level, were performed to select a the most appropriate functional monomer (FM) and porogenic solvent for the synthesis of the molecularly imprinted polymers (MIP). For the development of a water-compatible MIP chlorothiazide, hydrochlorothiazide or hydrfoflumethiazide were used as template, acrylamide as FM, ethyleneglycol dimethacrylate as cross-linker and tetrahydrofuran as porogenic solvent. Parameters that affect thepolymer efficiency, such as the amount of the monomer and nature of the cross-linker were evaluated. The adsorption isotherms for each of the synthesized polymers were constructed and the selectivities of the MIPs in relation to structural analogues of the templates were evaluated. The polymers were characterized by Fourier transform infrared, 13C NMR, scanning electron microscopy, nitrogen sorption porosimetry and thermogravimetric analysis. The MIPs were employed as stationary phase in SPE for the determination of thiazide diuretics in urine by high performance liquid chromatography and they showed cross-selectivity in relation to their structural analogues / Doutorado / Quimica Analitica / Doutor em Ciências

Les antibiotiques sont les médicaments les plus utilisés dans la médecine moderne. Depuis leurs découvertes, ils ont drastiquement changé la façon dont les infections sont traitées. Toutefois, à travers le processus d’adaptation, les bactéries deviennent éventuellement résistantes aux antibiotiques. Malgré leur omniprésence dans la biosphère, l’émergence de souches résistantes est favorisée par le mauvais usage des antibiotiques, ce qui crée une menace importante pour la santé publique. Les antibiotiques actuelles perdent graduellement leur efficacité, et vue le faible nombre de nouvelles molécules développées, la priorité est donnée pour la découverte de nouvelles stratégies capables de combattre les pathogènes. Les nouvelles cibles thérapeutiques idéales doivent exercer une faible pression évolutive, diminuer la virulence et être unique aux microorganismes. Une façon d’atteindre cet objectif est d’interférer dans la régulation et l’homéostasie du Fer chez les bactéries. La biodisponibilité du Fer a fortement influencé l’émergence de la vie sur terre et les stratégies évolutives qu’elle a adoptée. Ce qui a mené à l’apparition d’un mécanisme central de détection du Fer assurant la régulation de cet élément de haute importance. Ce senseur est un point faible que nous pourrons exploiter dans notre combat contre les infections bactériennes. La protéine Fur, pour « Ferric Uptake Regulator », est un régulateur de transcription métal dépendant qui est impliqué dans vaste réseau de régulation contrôlant principalement l’homéostasie du Fer et l’expression de facteurs de virulence. Le travail présenté dans ce manuscrit complète les études précédentes sur des inhibiteurs de la protéine Fur en utilisant une approche combinée théorique et expérimentale grâce a des expériences de XAS, SAXS et MALLS associé a de la modélisation moléculaire. Nous décrivons pour la première fois la structure de Fur d’E. coli ainsi que la structure d’un tétramère de Fur d’un mutant de P. aeruginosa. Par ailleurs, les profils d’énergie libre des protéines Fur de différentes espèces ont été déterminé, pour des complexes tetramériques ou dans le cas de dimères liés à l’ADN, permettant une compréhension préliminaire de leur mécanistique. Les informations structurales obtenues grâce aux travaux présentés dans ce manuscrit permettront de mieux comprendre les mécanismes d’inhibition des protéines Fur ainsi que fournir de nouvelles opportunités pour le développement de molécules a visée thérapeutique. / The most commonly prescribed drugs in human medicine are antibiotics. Since their discovery, they have drastically impacted the way we treat infections. However, a bacterium eventually becomes resistant to antimicrobial treatment through the natural process of adaptative evolution. Even if resistant bacteria are omnipresent in the biosphere, their emergence rate is accelerated by the misuse of antimicrobial agents leading to the public health threat we are facing now. As currently available antimicrobial agents lose their effectiveness and very few new drugs are being developed, a breakthrough in new strategies to fight pathogens should be a priority. Ideal new therapeutic targets should exert weak evolutionary pressure, disarm or weaken the pathogen and be unique to microorganisms. One way to do so is by interfering with the iron regulation and its homeostasis within Bacteria. The bioavailability of iron strongly influenced early life and the metabolic strategies that sustained it. A central iron sensing mechanism evolved to ensure the regulation of such an important element. Sadly for bacteria this sensor became an exploitable weakness in our battle against infection. The “Ferric Uptake Regulator” is a metal dependent transcription regulator with a large regulatory network controlling iron homeostasis and bacterial virulence. This work continues previous investigations on Fur inhibitors using a combined experimental and theoretical approach by performing XAS, SAXS and MALLS experiments together with computer simulations. We describe for the first time the structures of Fur from E. coli in addition to a tetrameric Fur structure of a mutant from P. aeruginosa. Moreover, free energy profiles of Fur proteins, as tetramers or dimers bound to DNA, from different species were generated and key residues involved in the interactions determined, providing mechanistic insights into Fur complexes. The structural information gathered from this work will be used to better understand inhibition mechanisms of Fur proteins providing new opportunities to overcome drug development challenges.

Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-β1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-β1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-β1 to its TGF-β receptor-I (TβR-I) and -II (TβR-II). However, sequential addition of sHA3 to the TβR-II/TGF-β1 complex led to a significantly stronger recruitment of TβR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TβR-I when added sequentially. For the first time bioactivity of TGF-β1 in conjunction with sHA was investigated at the receptor level. TβR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-β1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-β1-driven skin fibrosis.

This master's thesis focuses on search and structure prediction of quadruplexes in DNA sequences. Thesis also explains related terms that are important for understanding the function, properties and geometry of quadruplexes. Thesis describe physico-chemical and computational current methods, which possible to discover and structure prediction. This paper also explain the principle of molecular modelling, which was used in the final application. Design and implementation of the final algorithm are also part of this thesis.