Made available in DSpace on 2017-07-24T19:38:12Z (GMT). No. of bitstreams: 1 VivianePaula.pdf: 3188050 bytes, checksum: 1b1ca9983470b6e6a3669cfd52a8c846 (MD5) Previous issue date: 2007-03-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The enzymes dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase (PTR) are involved in the pterin/folate dependent metabolism; together they represent an important target for chemotherapy of parasitic leishmanias and trypanosomes. Xray crystallography was used to elucidate accurately the structure of the PTR1 enzyme from Trypanosoma brucei in complex with inhibitors which are analogous to the substrate. The ligands assayed for crystallization were the substrate folate and the inhibitors melamine, 6-thioguanine, WSG1012, WSG1034, WSG3065, WSG3066 and WSG3067. Of these, four yielded crystals with diffraction patterns sufficient for a complete dataset. WSG3065 (later revealing the lack of the ligand), WSG3066 and WSG3067 are three of the several structures presented in this work which came from the cited crystallization assays; added to these are the refined structures complexed with triamterene and cyromazine, proceeded from two other datasets already available. The datasets were processed with the programs Mosflm / Scala and Xds / Xscale, the structures were refined using the programs CNS and Refmac5 and validated with the programs Procheck, Whatcheck, Sfcheck and ValidationPDB. All refined structures belong to the space group P21 with unit cells around a = 79, b = 90, c = 82, b = 115, 4 monomers each of 268 residues per asymmetric unit and complex active sites. Besides the inhibiting ligands (except WSG3065) present in the structure, other ligands were found either near or outside the active site: dithiothreitol, glycerol, ethylene glycol, sodium and acetate ions. Analyses on the ligand positions and corresponding interactions with the protein were carried out to understand modes of inhibition and to guide the design or the discovery of new compounds which are potent, but selective to the parasitic enzyme, inhibitors. Thereby, initial docking studies were performed aiming at identifying new molecules or lead compounds with inhibitory capabilities. / As enzimas dihidrofolato redutase-timidilato sintase (DHFR-TS) e pteridina redutase (PTR) estão envolvidas no metabolismo pterina/folato dependente; juntas, representam um importante alvo para a quimioterapia de leishmanias e tripanossomas parasitas. A Cristalografia por Raios X foi utilizada para elucidar acuradamente a estrutura da enzima PTR1 de Trypanosoma brucei complexada com inibidores que são análogos ao substrato. Os ligantes ensaiados para cristalização foram o substrato folato e os inibidores melamina, 6-tioguanina, WSG1012, WSG1034, WSG3065, WSG3066 e WSG3067. Destes, quatro forneceram cristais com padrões de difração suficientes para um conjunto de dados completo. WSG3065 (mais tarde revelando ausência do ligante), WSG3066 e WSG3067 são três das estruturas apresentadas neste trabalho derivadas dos ensaios de cristalização citados; somadas a estas estão as estruturas refinadas dos complexos com triantereno e ciromazina, provenientes de dois outros conjuntos de dados anteriormente disponíveis. Os conjuntos de dados foram processados com os programas Mosflm / Scala e Xds / Xscale, as estruturas refinadas usando-se os programas CNS e Refmac5 e validadas com os programas Procheck, Whatcheck, Sfcheck e ValidationPDB. Todas as estruturas refinadas apresentaram grupo espacial P21 com celas unitárias aproximadas a = 79 = 90, c = 82 , b = 115, 4 monômeros de 268 resíduos cada por unidade assimétrica e sítios ativos complexos. Além dos ligantes inibidores presentes nas estruturas (exceto WSG3065), outros ligantes foram encontrados próximos ou fora do sítio ativo: ditiotreitol, glicerol, etilenoglicol, íons sódio e íons acetato. Análises das posições dos ligantes inibidores e correspondentes interações com a proteína foram realizadas a fim de se entender modos de inibição e, em particular, assistir ao planejamento ou à descoberta de novos compostos que sejam inibidores potentes, mas seletivos, para a enzima parasitária. Assim, estudos iniciais de atracagem (docking) foram realizados visando identificar novas moléculas ou arcabouços com capacidades inibitórias.

La dihydrofolate réductase humaine (DHFRh) est une enzyme essentielle à la prolifération cellulaire. Elle réduit le dihydrofolate en tétrahydrofolate, un co-facteur impliqué dans la biosynthèse des purines et du thymidylate. La DHFRh est une cible de choix pour des agents de chimiothérapie comme le méthotrexate (MTX), inhibant spécifiquement l’enzyme ce qui mène à un arrêt de la prolifération et ultimement à la mort cellulaire. Le MTX est utilisé pour le traitement de plusieurs maladies prolifératives, incluant le cancer. La grande utilisation du MTX dans le milieu clinique a mené au développement de mécanismes de résistance, qui réduisent l’efficacité de traitement. La présente étude se penche sur l’un des mécanismes de résistance, soit des mutations dans la DHFRh qui réduisent son affinité pour le MTX, dans le but de mieux comprendre les éléments moléculaires requis pour la reconnaissance de l’inhibiteur au site actif de l’enzyme. En parallèle, nous visons à identifier des variantes plus résistantes au MTX pour leur utilisation en tant que marqueurs de sélection en culture cellulaire pour des systèmes particuliers, tel que la culture de cellules hématopoïétiques souches (CHS), qui offrent des possibilités intéressantes dans le domaine de la thérapie cellulaire. Pour étudier le rôle des différentes régions du site actif, et pour vérifier la présence d’une corrélation entre des mutations à ces régions et une augmentation de la résistance au MTX, une stratégie combinatoire a été dévelopée pour la création de plusieurs banques de variantes à des résidus du site actif à proximité du MTX lié. Les banques ont été sélectionnées in vivo dans un système bactérien en utilisant des milieux de croissance contenant des hautes concentrations de MTX. La banque DHFRh 31/34/35 généra un nombre considérable de variantes combinatoires de la DHFRh hautement résistantes au MTX. Les variantes les plus intéressantes ont été testées pour leur potentiel en tant que marqueur de sélection dans plusieurs lignées cellulaires, dont les cellules hématopoïétiques transduites. Une protection complète contre les effets cytotoxiques du MTX a été observée chez ces cellules suite à leur infection avec les variantes combinatoires. Pour mieux comprendre les causes moléculaires reliées à la résistance au MTX, des études de structure tridimensionnelle de variantes liées au MTX ont été entreprises. La résolution de la structure de la double variante F31R/Q35E lié au MTX a révélé que le phénotype de résistance était attribuable à d’importantes différences entre le site actif de la double variante et de l’enzyme native, possiblement dû à un phénomème dynamique. Une compréhension plus générale de la reconnaissance et la résistance aux antifolates a été réalisée en comparant des séquences et des structures de variantes de la DHFR résistants aux antifolates et provenant de différentes espèces. En somme, ces travaux apportent de nouveaux éléments pour la comprehension des intéractions importantes entre une enzyme et un ligand, pouvant aider au développement de nouveaux antifolates plus efficaces pour le traitement de diverses maladies. De plus, ces travaux ont généré de nouveaux gènes de résistance pouvant être utilisés en tant que marqueurs de sélection en biologie cellulaire. / Human dihydrofolate reductase (hDHFR) is an enzyme that is essential to cell proliferation. It reduces dihydrofolate to tetrahydrofolate, an important cofactor involved in purine and thymidylate biosynthesis. hDHFR is a choice target for chemotherapeutic drugs like methotrexate (MTX), which specifically inhibits the enzyme, stopping cell proliferation and leading to cellular death. MTX is used for the treatment of many proliferative diseases, including cancers. Widespread use of MTX has lead to the development of resistance mechanisms appear which impair treatment efficiency. The present work focuses on a mechanism of resistance, namely mutations in hDHFR that reduce its affinity for MTX, to better understand the underlying mechanisms of inhibitor recognition at the active site of the enzyme. In parallel, we aim at identifying the most MTX-resistant variants to offer novel selectable markers for particular cell culture systems, such as hematopoietic cell culture, which offer important perspectives for cellular therapy. To study the role of different regions of the hDHFR active site, and to verify if a correlation exists between mutations in these regions and increased resistance to MTX, a combinatorial strategy was developed enabling the creation of several hDHFR variant libraries at active site residues located in proximity to bound MTX. The libraries were selected in vivo in a bacterial system using culture media containing high concentration of the inhibitor. One library in particular, hDHFR 31/34/35, yielded a considerable number of highly MTX-resistant combinatorial hDHFR variants. The most interesting candidates were tested for their potential as selectable markers in various cell lines, including transduced hematopoietic cells. Complete protection from MTX-cytotoxicity was obtained for these cells following infection with the combinatorial variants. To better understand the molecular causes of MTX resistance, resolution of the crystal structures of variant proteins in presence of MTX was attempted. Resolution of the F31R/Q35E double variant revealed that the resistance phenotype was related to important differences in the active site relative to WT, possibly attributable to a dynamic motion effect. A more general comprehension of antifolate recognition and resistance was achieved by sequence and structural comparison of antifolate-resistant DHFR variants from different species. Overall, our work contributes to the better understanding of enzyme-inhibitor interactions, which could provide new insights into the development of more efficient clinical therapies. In addition, this work has yielded novel drug-resistance genes useful as selectable markers for cellular biology.

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