Protein motions are complex, including occurring at different time scales, and their roles in enzyme-catalyzed reactions have always been of great interest among enzymologists. In order to characterize the potential factors that play a role on the chemical step of enzymatic reactions, variants of dihydrofolate reductase have been used as a benchmark system to study the motions of proteins correlated with the chemical step. A “global dynamic network” of coupled residues in Escherichia coli dihydrofolate reductase (ecDHFR), which assists in catalyzing the chemical step, has been demonstrated through quantum mechanical/molecular mechanical and molecular dynamic (QM/MM/MD) simulations, as well as bioinformatic analyses. A few specific residues — M42, G121, and I14 — were shown to function synergistically with measurements of single turnover rates and the temperature dependence of intrinsic kinetic isotope effects (KIEsint) of site-directed mutants. Although similar networks have been found in other enzymes, the general features of these networks are still unclear. This project focuses on exploring homologous residues of the proposed global network in human DHFR through computer simulations and measurements of the temperature dependence of KIEsint. The mutants M53W and S145V, both remote residues, showed significant decreases in catalytic efficiency. Non-additive isotope effects on activation energy were observed between M53 and S145, indicating their synergistic effect on hydride transfer in human DHFR. Apart from the effects of the conserved residues, we also extend our studies to exploring three potential phylogenetic events that account for the discrepancies between E. coli and human DHFR. They are L28, PP insertion and PEKN insertions by phylogenetic sequence analysis. Two of them (N23PP and G51PEKN E. coli DHFR) have been proved to be important both by MD simulation and experimental probe of KIEs measurement. The experiments have found that PP insertion itself rigidified the M20 loop and motions coupled to hydride transfer were impaired, however, loop rigidification was improved after incorporating PEKN. Furthermore, deletion of PP and PEKN of the engineered human enzyme also show a similar outcome. However, the effect of the key residue of L28 is not clear. In this project, we have step-wise engineered the human DHFR to be like hagfish (F31M) and E. coli (F32L). And it is found out that there is an increase in the temperature dependence of KIEs when the enzyme was bacterilized into a more primitive variant. This indicates that not only is residue F32 important and correlated with the chemical step as indicated by bioinformatic studies, but it is possible to trace the evolutionary trajectory. A triple mutation F32L-PP26N-PEKN62G on the human DHFR was also conducted, and it is not surprising to find out that the temperature dependence of KIEs has retained its behavior like wild-type human DHFR. These results suggest that the three predicted phylogenetically coherent events coevolved together to maintain the evolutionary preservation of the protein dynamics to enable H-tunneling from well-reorganized active sites. As has been indicated by the previous project, as the enzyme evolves, the active site of the enzyme will “reorganize” to form the optimal transition state for chemical step (from F32L-F32M-wild type DHFR). Here in this project, we aimed to systematically address this point of view through a series of cyclic permutation DHFR from directed evolutions. As this primitive enzyme is 7 orders of magnitude less efficient than the well-evolved human DHFR, together with four generations of evolved variants (cp, cp’ and cp”), this provides a good model system for explorations of the molecular basis of enzyme evolution. It is found that the organizations of transition state are improved before the catalytic efficiency is enhanced as the enzyme evolves.

<p>The main objective of the research described in this thesis has been the design and synthesis of inhibitors of the enzyme dihydrofolate reductase (DHFR) intended for local administration and devoid of systemic side-effects. The blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infections, and also opportunistic infections associated with AIDS (<i>Pneumocystis carinii</i> pneumonia, PCP). Recent research indicates that the enzyme also is involved in various autoimmune diseases, e.g., rheumatoid arthritis, inflammatory bowel diseases and psoriasis. Many useful antifolates have been developed to date although problems remain with toxicity and selectivity, e.g., the well-established, classical antifolate methotrexate exerts a high activity but also high toxicity. The new antifolates described herein were designed to retain the pharmacophore of methotrexate, but encompassing an ester group, so that they also would serve as substrates for the endogenous hydrolytic enzymes, e.g., esterases. Such antifolates would optimally comprise good examples of <i>soft drugs</i> because they in a controlled fashion would be rapidly and predictably metabolized to non-toxic metabolites after having exerted their biological effect at the site of administration.</p><p>A preliminary screening of a large series of simpler aromatic esters as model compounds in a biological assay consisting of esterases from different sources was performed. The structural features of the least reactive ester were substituted for the methyleneamino bridge in methotrexate to produce analogues that were chemically stable but potential substrates for DHFR as well as for the esterases.</p><p>The new inhibitor showed desirable activity towards rat liver DHFR, being only eight times less potent then methotrexate. Furthermore, the derived metabolites were found to be poor substrates for the same enzyme. The new compound showed good activity in a mice colitis model <i>in vivo</i>, but a pharmacokinetic study revealed that the half-life of the new compound was similar to methotrexate. A series of compounds characterized by a high lipophilicity and thus expected to provide better esterase substrates were designed and synthesized. One of these analogues in which three methoxy groups were substituted for the glutamic residue of methotrexate exhibited favorable pharmacokinetics. This compound is structurally similar to another potent DHFR inhibitor, trimetrexate, used in the therapy of PCP (<i>vide supra</i>). The new inhibitor that undergoes a fast metabolism <i>in vivo</i> is suitable as a model to further investigate the soft drug concept.</p>

Malaria is a life-threatening parasitic disease that causes at least 300 million acute illnesses annually, of which at least one million infected humans die, mainly children under the age of 5 years. This overwhelming burden is due to the most virulent causative agent, Plasmodium falciparum, as a result of its prevalence in sub-Saharan Africa, as well as its resistance to nearly all anti-malarials in use. There is thus an urgent need to discover and characterise new novel parasitic targets for chemotherapeutic intervention. Folate metabolism is the target of several anti-malarials such as pyrimethamine and sulfadoxine. These drugs cause a decrease in parasite growth since Plasmodia have a high rate of replication and demand for nucleotides during DNA synthesis. The parasite is almost totally resistant to the current antifolates. Further insights into the folate pathway and its drugs are essential for the understanding of the resistance mechanism and to identify/characterise new drug targets for inhibition. Three possible new drug targets were identified and characterised in the folate pathway (Lee C.S. et al., 2001). One of these targets is the bifunctional enzyme, dihydrofolate-synthase folylpolyglutamate synthase (DHFS-FPGS). The bifunctionality and activity of the -dhfs-fpgs gene in Plasmodium was confirmed by functional complementation in yeast and bacteria and shown to be unique to Plasmodia and bacteria. This gene indicated only a 15-17% homology to other organisms; DHFS activity is usually only present in prokaryotes but not in humans or other eukaryotes (Salcedo E. et al., 2001). Although part of a bifunctional protein and having closely related catalytic functions, the DHFS and FPGS activities have distinct roles to play in both the de novo and salvage pathways of folate metabolism. These characteristics indicate DHFS-FPGS as a potentially good drug target since a single inhibitor is likely to have a drastic effect on both routes and consequently arrest DNA synthesis in the malaria parasite. This could prove to be a very effective and novel antimalarial strategy. Comparative expression studies of synthetic and native DHFS-FPGS presented here indicated that the highest quantity of protein is expressed from the synthetic gene. However, results indicated that most of the recombinant protein expressed in various E. coli cell lines, produced insoluble protein aggregates. Various strategies were employed in an attempt to improve recombinant soluble expression including auto-induction of T7 promoter activity. However, this did not result in an increased percentage of soluble protein expression even though improved total protein expression was observed. The inclusion of chaperone proteins resulted in a minor change in soluble expression. Activity assays of the DHFS-FPGS from these two methods indicated that active protein was produced in a correctly folded manner. Due to the high amount of recombinant protein present in the inclusion bodies, various methods were investigated to isolate and refold the DHFS-FPGS protein. The use of a non-ionic and ionic detergent for refolding resulted in pure, solubilised, active protein. Activity assays of the refolded, soluble protein indicated that the protein is active. Preliminary kinetic analysis was unsuccessful and requires further investigations. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

Le dihydrofolate réductase (DHFR) est la principale cible du méthotrexate, un important composant du traitement de la leucémie lymphoblastique aiguë (LLA). Une association des polymorphismes du promoteur de DHFR avec l’issue de la LLA a été mise en évidence au laboratoire. Une survie sans événement (EFS) réduite corrélait avec les allèles A -317 et C -1610, et l’haplotype *1, défini par ces allèles. L’haplotype *1 était aussi associé à une expression élevée du DHFR. Dans cette étude, nous étendons l’analyse à la région régulatrice adjacente, d’environ 400 pb, correspondant au transcrit mineur non-codant du DHFR, qui joue un rôle essentiel dans la régulation de la transcription au niveau du promoteur majeur. Six polymorphismes ont été identifiés, parmi lesquels 5 étaient des SNPs et un polymorphisme de longueur composé d’un nombre variable d’éléments de 9 pb et d’une insertion/délétion de 9 pb. L’analyse d’haplotype, incluant tous les polymorphismes promoteurs, a révélé une diversification de l’haploytpe *1 en 5 sous-types (*1a à *1e). Les variations du promoteur majeur et les sous-types de l’haplotype *1 ont été par la suite analysés pour l’association avec l’issue de LLA. Un EFS réduit corrélait avec l’allèle A du polymorphisme G308A (p=0,02) et avec l’haplotype *1 (p=0,01). Des niveaux élevées d’ARNm étaient trouvés chez les porteurs de l’haplotype *1b (p=0,005) et pas pour les autres sous-types de l’haplotype *1. Alors, la mauvaise issue de LLA associée avec l'haplotype *1 est en effet déterminée par le sous-type *1b. Cette étude donne un nouvel aperçu des polymorphismes régulateurs du DHFR définissant plus précisément les variations du DHFR prédisposant un événement. / Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event free survival (EFS) correlated with the alleles A -317 and C -1610, and with haplotype *1, defined by these alleles. Haplotype*1 was also associated higher DHFR expression. Here we extended the analysis to adjacent 400bp regulatory region corresponding to non-coding minor DHFR transcript which plays an essential role in the regulation of transcription from the major promoter. Six polymorphisms were identified, of which 5 were SNPs and one length polymorphism composed of variable number of 9bp elements and 9bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into 5 subtypes (*1a to *1e). Major promoter variations and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower EFS correlated with an A allele of G308A polymorphism (p=0.02) and with *1b haplotype (p=0.01). Higher mRNA levels were found in the carriers of *1b haplotype (p=0.005) and not for remaining haplotype *1 subtypes. So, the worse ALL outcome associated with haplotype *1 is actually determined by the subtype *1b. The study provides a new insight into DHFR regulatory polymorphisms defining more precisely event–predisposing DHFR variations.

No description available.

Le triméthoprime (TMP) est un antibiotique communément utilisé depuis les années 60. Le TMP est un inhibiteur de la dihydrofolate réductase (DHFR) bactérienne chromosomale. Cette enzyme est responsable de la réduction du dihydrofolate (DHF) en tétrahydrofolate (THF) chez les bactéries, qui lui, est essentiel à la synthèse des purines et ainsi, à la prolifération cellulaire. La résistance bactérienne au TMP est documentée depuis plus de 30 ans. Une des causes de cette résistance provient du fait que certaines souches bactériennes expriment une DHFR plasmidique, la DHFR R67. La DHFR R67 n'est pas affectée par le TMP, et peut ainsi remplacer la DHFR chromosomale lorsque celle-ci est inhibée par le TMP. À ce jour, aucun inhibiteur spécifique de la DHFR R67 est connu. En découvrant des inhibiteurs contre la DHFR R67, il serait possible de lever la résistance au TMP que la DHFR R67 confère aux bactéries. Afin de découvrir des inhibiteurs de DHFR R67, les approches de design à base de fragments et de criblage virtuel ont été choisies. L'approche de design à base de fragments a permis d'identifier sept composés simples et de faible poids moléculaire (fragments) inhibant faiblement la DHFR R67. À partir de ces fragments, des composés plus complexes et symétriques, inhibant la DHFR R67 dans l'ordre du micromolaire, ont été élaborés. Des études cinétiques ont montré que ces inhibiteurs sont compétitifs et qu'au moins deux molécules se lient simultanément dans le site actif de la DHFR R67. L'étude d'analogues des inhibiteurs micromolaires de la DHFR R67 a permis de déterminer que la présence de groupements carboxylate, benzimidazole et que la longueur des molécules influencent la puissance des inhibiteurs. Une étude par arrimage moléculaire, appuyée par les résultats in vitro, a permis d'élaborer un modèle qui suggère que les résidus Lys32, Gln67 et Ile68 seraient impliqués dans la liaison avec les inhibiteurs. Le criblage virtuel de la librairie de 80 000 composés de Maybridge avec le logiciel Moldock, et les essais d'inhibition in vitro des meilleurs candidats, a permis d'identifier quatre inhibiteurs micromolaires appartenant à des familles distinctes des composés précédemment identifiés. Un second criblage virtuel, d'une banque de 6 millions de composés, a permis d'identifier trois inhibiteurs micromolaires toujours distincts. Ces résultats offrent la base à partir de laquelle il sera possible de développer iv des composés plus efficaces et possédant des propriétés phamacologiquement acceptables dans le but de développer un antibiotique pouvant lever la résistance au TMP conféré par la DHFR R67. / Trimethoprim (TMP) is a common antibiotic which is used since the 60's. TMP is an inhibitor of the bacterial chromosomal dihydrofolate reductase (DHFR). This enzyme catalyses the reduction of the dihydrofolate (DHF) to tetrahydrofolate (THF) which is essential to the biosynthesis of purines thus to cellular proliferation. Bacterial TMP resistance is documented since about 30 years. One of the cause of this resistance comes from the fact that certain bacteria express a plasmidic DHFR, the R67 DHFR, which confers TMP resistance. The R67 DHFR is not inhibited by TMP and can replace the chromosomal DHFR when the latter is inhibited by TMP. The discovery of R67 DHFR inhibitors would allow to break the trimethoprim resistance granted by R67 DHFR. In order to discover R67 DHFR inhibitors, fragment based design and virtual screening approaches were selected. By fragment based design, seven simple compounds with a low molecular mass which inhibited weakly R67 DHFR (fragments) were identified. From these fragments, more complex and symmetrical compounds inhibiting R67 DHFR in the micromolar range were identified. Kinetic studies showed these inhibitors were competitive and at least two molecules bind simultaneously to the active site of the R67 DHFR. Test of the micromolar inhibitors analog showed that the presence of carboxylate, benzimidazole and the length of the molecule all have an effect on the potency of the inhibitors. Molecular docking of the inhibitors, supported by in vitro data, were used to develop a model which suggest that residue like Lys32, Gln67 and Ile68 would be involved in the binding of the inhibitors to the R67 DHFR. Virtual screening of the 80 000 compound Maybridge library with Moldock software, followed by in vitro test of the best candidate, identified four micromolar inhibitors which are chemically distinct from the inhibitor beforehand identified. A second virtual screening of a 6 million compounds bank identified three micromolar inhibitors which are also distinct from the inhibitor beforehand identified. vi These results offer a basis which will allow further development of more potent inhibitors with more acceptable pharmacologic properties in order to develop an antibiotic which would break the TMP resistance granted by the R67 DHFR.

Le dihydrofolate réductase (DHFR) est la principale cible du méthotrexate, un important composant du traitement de la leucémie lymphoblastique aiguë (LLA). Une association des polymorphismes du promoteur de DHFR avec l’issue de la LLA a été mise en évidence au laboratoire. Une survie sans événement (EFS) réduite corrélait avec les allèles A -317 et C -1610, et l’haplotype *1, défini par ces allèles. L’haplotype *1 était aussi associé à une expression élevée du DHFR. Dans cette étude, nous étendons l’analyse à la région régulatrice adjacente, d’environ 400 pb, correspondant au transcrit mineur non-codant du DHFR, qui joue un rôle essentiel dans la régulation de la transcription au niveau du promoteur majeur. Six polymorphismes ont été identifiés, parmi lesquels 5 étaient des SNPs et un polymorphisme de longueur composé d’un nombre variable d’éléments de 9 pb et d’une insertion/délétion de 9 pb. L’analyse d’haplotype, incluant tous les polymorphismes promoteurs, a révélé une diversification de l’haploytpe *1 en 5 sous-types (*1a à *1e). Les variations du promoteur majeur et les sous-types de l’haplotype *1 ont été par la suite analysés pour l’association avec l’issue de LLA. Un EFS réduit corrélait avec l’allèle A du polymorphisme G308A (p=0,02) et avec l’haplotype *1 (p=0,01). Des niveaux élevées d’ARNm étaient trouvés chez les porteurs de l’haplotype *1b (p=0,005) et pas pour les autres sous-types de l’haplotype *1. Alors, la mauvaise issue de LLA associée avec l'haplotype *1 est en effet déterminée par le sous-type *1b. Cette étude donne un nouvel aperçu des polymorphismes régulateurs du DHFR définissant plus précisément les variations du DHFR prédisposant un événement. / Dihydrofolate reductase (DHFR) is the major target of methotrexate, a key component in childhood acute lymphoblastic leukemia (ALL) treatment. We recently reported an association of DHFR promoter polymorphisms with ALL outcome. Lower event free survival (EFS) correlated with the alleles A -317 and C -1610, and with haplotype *1, defined by these alleles. Haplotype*1 was also associated higher DHFR expression. Here we extended the analysis to adjacent 400bp regulatory region corresponding to non-coding minor DHFR transcript which plays an essential role in the regulation of transcription from the major promoter. Six polymorphisms were identified, of which 5 were SNPs and one length polymorphism composed of variable number of 9bp elements and 9bp insertion/deletion. Haplotype analysis including all promoter polymorphisms revealed diversification of haplotype *1 into 5 subtypes (*1a to *1e). Major promoter variations and haplotype *1 subtypes were subsequently analyzed for the association with ALL outcome. Lower EFS correlated with an A allele of G308A polymorphism (p=0.02) and with *1b haplotype (p=0.01). Higher mRNA levels were found in the carriers of *1b haplotype (p=0.005) and not for remaining haplotype *1 subtypes. So, the worse ALL outcome associated with haplotype *1 is actually determined by the subtype *1b. The study provides a new insight into DHFR regulatory polymorphisms defining more precisely event–predisposing DHFR variations.

Le projet de recherche a été réalisé en collaboration avec Professeur Jean-François Masson du Département de Chimie de l'Université de Montréal. The research project was made in collaboration with Professor Jean-François Masson from the Chemistry department of University of Montreal. / L'objectif du projet de recherche est de développer une méthode de criblage d’inhibiteurs basée sur une technologie émergente, soit un dispositif portatif utilisant la résonance des plasmons de surface (SPR). La cible du criblage est la dihydrofolate réductase R67 (DHFR R67), une enzyme qui confère une résistance bactérienne à l'antibiotique triméthoprime. Ici, l'enzyme cible est immobilisée sur une surface d'or mince avec des propriétés plasmoniques (optiques) spécifiques qui varient en fonction de la masse des molécules se liant à cette surface. Cette technique permet de suivre les événements de liaison de molécules à la DHFR R67 immobilisée, et ainsi peut permettre l'identification d'inhibiteurs potentiels. Cependant, la masse molaire des inhibiteurs typiquement utilisés lors de criblages préliminaires (i.e. 500-1000 g/mol) est trop faible pour générer un signal SPR détectable. Afin de contrer cette lacune, ce mémoire a pour objet de développer un essai compétitif indirect qui mettra en jeu des molécules de masse supérieure. D’abord, une nanoparticule d'or portant un analogue de substrat se liera à la DHFR R67 immobilisée à la surface d’or, générant ainsi un signal SPR important en raison de la masse molaire élevée de la nanoparticule. Ensuite, lors du criblage d'inhibiteurs potentiels, les nanoparticules liées seront déplacées de l'enzyme cible si la molécule criblée fournit une affinité suffisante. Ainsi, il sera possible de suivre indirectement la liaison d'un inhibiteur à la cible. Ce projet vise donc à tester et à valider l'approche de criblage SPR appliquée à la DHFR R67. / The objective of the research project is to develop a method for inhibitor screening based on a portable Surface Plasmon Resonance (SPR) device, an emerging technology. The target is R67 dihydrofolate reductase (R67 DHFR), an enzyme that confers bacterial resistance to the antibiotic trimethoprim. Here, the target enzyme is linked to a thin gold surface having specific plasmonic (optical) properties that vary as a function of the mass of bound molecules. This allows monitoring binding to the surface-linked R67 DHFR, and thus permits identification of inhibitors. However, the mass of the low-affinity inhibitors typically identified in early stages of screening (i.e. 500-1000 g/mol) is too low to produce a significant SPR signal. To address this shortcoming, a competitive assay will be developed: a gold nanoparticle carrying a substrate analog will bind the surface-immobilized R67 DHFR, resulting in a strong SPR signal due to its high mass. Then, upon screening for potential inhibitors, the bound nanoparticle will be displaced from the target enzyme if a molecule provides sufficient affinity. By those means, it will be possible to indirectly monitor the binding of an inhibitor to the target. This goal of this project is to test and validate the SPR screening approach applied to R67 DHFR.

Although the tremendous catalytic power of enzymes is widely recognized, their exact mechanisms of action are still a source of debate. In order to elucidate the origin of their power, it is necessary to look at individual residues and atoms, and establish their contribution to ligand binding, activation, and reaction. Given the present limitations of experimental techniques, only computational tools allow for such detailed analysis. During my PhD studies I have applied a variety of computational methods, reviewed in Chapter 2, to the study of two enzymes: DfrB dihydrofolate reductase (DHFR) and methyltetrahydrofolate: corrinoid/iron-sulfur protein methyltransferase (MeTr). ¶ The DfrB enzyme has intrigued microbiologists since it was discovered thirty years ago, because of its simple structure, enzymatic inefficiency, and its insensitivity to trimethoprim. This bacterial enzyme shows neither structural nor sequence similarity with its chromosomal counterpart, despite both catalysing the reduction of dihydrofolate (DHF) using NADPH as a cofactor. As numerous attempts to obtain experimental structures of an enzyme ternary complex have been unsuccessful, I combined docking studies and molecular dynamics simulations to produce a reliable model of the reactive DfrB•DHF•NADPH complex. These results, combined with published empirical data, showed that multiple binding modes of the ligands are possible within DfrB. ¶ Comprehensive sequence and structural analysis provided further insight into the DfrB family. The presence of the dfrB genes within integrons and their level of sequence conservation suggest that they are old structures that had been diverging well before the introduction of trimethoprim. Each monomer of the tetrameric active enzyme presents an SH3-fold domain; this is a eukaryotic auxiliary domain never found before as the sole domain of a protein, let alone as the catalytic one. Overall, DfrB DHFR seems to be a poorly adapted catalyst, a ‘minimalistic’ enzyme that promotes the reaction by facilitating the approach of the ligands rather than by using specific catalytic residues. ¶ MeTr initiates the Wood-Ljungdahl pathway of anaerobic CO2 fixation. It catalyses the transfer of the N5-methyl group from N5-methyltetrahydrofolate (CH3THF) to the cobalt centre of a corrinoid/iron-sulfur protein. For the reaction to occur, the N5 position of CH3THF is expected to be activated by protonation. As experimental studies have led to conflicting suggestions, computational approaches were used to address the activation mechanism. ¶ Initially, I tested the accuracy of quantum mechanical (QM) methods to predict protonation positions and pKas of pterin, folate, and their analogues. Then, different protonation states of CH3THF and active-site aspartic residues were analysed. Fragment QM calculations suggested that the pKa of N5 in CH3THF is likely to increase upon protein binding. Further, ONIOM calculations which accounted for the complete protein structure indicated that active-site aspartic residues are likely to be protonated before the ligand. Finally, solvation and binding free energies of several protonated forms of CH3THF were compared using the thermodynamic integration approach. Taken together, these preliminary results suggest that further work with particular emphasis on the protonation state of active-site aspartic residues is needed in order to elucidate the protonation and activation mechanism of CH3THF within MeTr.

Made available in DSpace on 2016-08-17T18:39:34Z (GMT). No. of bitstreams: 1 3220.pdf: 7184046 bytes, checksum: d31437c1aa1937336c7b8cb91918b19b (MD5) Previous issue date: 2010-08-06 / Universidade Federal de Minas Gerais / Malaria disease, caused mainly by Plasmodium falciparum parasite, afflicts about 500 million people and causes nearly one million deaths every year. For the development of new drugs against this disease, one possible approach is to identify an enzyme that plays a key role in P. falciparum development and presents significantly different properties from the corresponding human one. These differences can be exploited in the design of specific inhibitors of the parasite s protein, thus, three different enzymes were selected as possible targets. As there are evidences suggesting that increasing oxidative stress can effectively inhibit the growth of the malarial parasite the enzyme Glutathione Reductase of P. falciparum (PfGR), responsible for the parasite s antioxidant defense, has become a potential target for the design and development of inhibitors. The second target was the P. falciparum Dihydrofolate Reductase-Thymidylate Synthase (PfDHFR-TS), and in this case blocking its action stops the dTMP production and DNA synthesis in the parasite. The third chosen target was the P. falciparum Lactate Dehydrogenase (PfLDH), whose inhibition interrupts the ATP formation and thus causing the death of the parasite. So that a family of arilloxazines compounds, together with chloroquine and methylene blue, were studied by means of docking simulations in the binding sites of these enzymes and also in the corresponding human enzymes for comparison. The three-dimensional structures of the enzymes and of chloroquine and methylene blue were obtained from the Protein Data Bank (PDB). The structures of the arilloxazines compounds, in turn, were obtained by molecular modeling with HyperChem 6.01 and MOPAC2009 programs, using as starting models similar crystallographic structures deposited in the Cambridge Structural Database. Docking simulations were performed using GOLD 4.0.1. The docking results showed that the enzymes PfGR and PfDHFR-TS are not the preferential targets of chloroquine. For the methylene blue it was possible to elucidate its binding mode in hGR and PfGR. Regarding the arilloxazines it was possible to show that they present their higher affinity for hGR, followed by PfGR, hDHFR, PfDHFR-TS, PfLDH and hLDH. In the case of GRs, the interface site was the preferred binding site. The results suggest that if arilloxazines compounds with higher affinity for PfGR are desirable then a pentafluorophenyl should be attached at the N10 position, as in the 2e compound. When searching for arilloxazines with higher affinity for PfLDH, it seems to be desirable a carboxymethyl group at the N3 position (as in 5b) and a pentafluorophenyl group at N10 (as in 2e). Finally, the results suggest that in general the studied arilloxazines probably will present a higher affinity for hDHFR than PfDHFR-TS. All these results are an important starting point for the design of new arilloxazines ligands so that they can be used as lead compounds in the search for new drugs against malaria. / A malária, causada principalmente pelo Plasmodium falciparum, atinge cerca de 500 milhões de pessoas e causa aproximadamente um milhão de mortes todos os anos. Para o desenvolvimento de novos fármacos contra esta doença, uma das abordagens possível é identificar uma enzima que desempenhe papel vital no desenvolvimento do P. falciparum e apresente propriedades significantemente diferentes das enzimas humanas correspondentes, de modo que tais diferenças possam ser exploradas no design de inibidores específicos à proteína do parasita. Existem evidências sugerindo que aumentar o estresse oxidativo pode inibir eficientemente o crescimento do parasita causador da malária e, portanto, a enzima Glutationa Redutase do P. falciparum (GRPf), responsável por sua defesa antioxidante, tornou-se um alvo em potencial para o desenvolvimento de inibidores. Também, o bloqueio da ação da Diidrofolato Redutase-Timidilato Sintase do P. falciparum (DHFR-TSPf) interrompe a produção de dTMP e a síntese de DNA no parasita. Ainda, espera-se que a inibição da Lactato Desidrogenase do P. falciparum (LDHPf) interrompa a produção de ATP no parasita e, consequentemente, cause sua morte. Portanto, estudou-se o comportamento de compostos da família das ariloxazinas, da cloroquina e do azul de metileno nos sítios de ligação destas enzimas, além das enzimas humanas correspondentes para fins de comparação, por meio de cálculos de docking. As estruturas tridimensionais das enzimas foram obtidas no Protein Data Bank (PDB). As estruturas dos inibidores da família das ariloxazinas, por sua vez, foram obtidas por meio de modelagem molecular, utilizando os programas HyperChem 6.01 e MOPAC2009, a partir de estruturas cristalográficas semelhantes obtidas no Cambridge Structural Database; já as estruturas da cloroquina e do azul de metileno foram obtidas também no PDB. Os cálculos de docking destes compostos nos sítios de ligação das enzimas estudadas foram realizados utilizando o programa GOLD 4.0.1. Com base nos resultados de docking, sugere-se que as enzimas GRPf e DHFR-TSPf não são alvos preferenciais da cloroquina. Também, pôde-se elucidar o possível modo de ligação do azul de metileno nas enzimas GRh e GRPf. No geral, foi possível sugerir ainda que as ariloxazinas devam apresentar maior afinidade pela GRh, seguida por GRPf, DHFRh, DHFR-TSPf, LDHPf e LDHh, nesta ordem. Nas GRs, o sítio da interface foi o sítio preferencial de ligação. Para se buscar inibidores da família das ariloxazinas com maior afinidade pela GRPf, sugere-se considerar um pentafluorfenil como substituinte na posição N10, como no composto 2e. Ainda, na busca por ariloxazinas com maior afinidade pela LDHPf, sugere-se considerar um carboximetil na posição N3 (como o de 5b) e um pentafluorfenil na posição N10 (como em 2e). Por fim, foi obtido que as ariloxazinas estudadas possivelmente apresentarão, em geral, uma maior afinidade pela DHFRh do que pela DHFR-TSPf. Estes dados podem ser tomados como ponto de partida para o design de novos compostos da família das ariloxazinas, a fim de que possam atuar como compostos líderes na busca por novos fármacos contra a malária.