Made available in DSpace on 2014-12-17T14:03:30Z (GMT). No. of bitstreams: 1 RodrigoOA.pdf: 1265658 bytes, checksum: 99f4f1b083a5b39748d0fb4e05fcf79f (MD5) Previous issue date: 2009-02-06 / Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane / A quitina (homopol?mero linear contendo res?duos de β-1,4-N-acetil-D-glicosamina (GlcNac) ? um importante componente estrutural da parede celular de fungos e exoesqueletos de muitos invertebrados pragas, tais como insetos e nemat?ides. Em sistemas digest?rios de insetos forma uma matriz denominada de membrana peritr?fica. Um dos mais estudados modelos de intera??o prote?na-carboidrato ? o modelo que envolve as prote?nas ligantes ? quitina. Dentre os motivos j? caracterizados envolvidos nesta intera??o se destacam o motivo heve?na (HD), obtida de Hevea brasiliensis (Seringueira), o motivo R&R consenso (R&R), encontrado em prote?nas cuticulares de insetos, e o motivo denominado neste estudo como motivo conglicinina (CD), encontrado na estrutura cristalogr?fica da β-conglicinina complexada com GlcNac. Estes tr?s motivos de liga??o ? quitina foram usados para determinar qual(is) deles poderia(m) estar envolvido(s) in silico na intera??o das vicilinas de Canavalia ensiformis e Vigna unguiculata com quitina, como tamb?m associar estes resultados com o WD50 destas vicilinas para larvas de Callosobruchus maculatus. A t?cnica de modelagem comparativa foi utilizada para constru??o do modelo 3D da vicilina de V. unguiculata, que n?o foi encontrada nos bancos de dados. Atrav?s do programa ClustalW obteve-se a localiza??o destes dom?nios na estrutura prim?ria das vicilinas. Os dom?nios R&R e CD foram encontrados com maior homologia nas seq??ncias prim?rias das vicilinas e foram alvos de estudos de intera??o. Atrav?s do programa GRAMM foram obtidos modelos de intera??o ( dockings ) das vicilinas com GlcNac. Os resultados mostraram que, atrav?s de an?lises in silico, o motivo HD n?o faz parte da estrutura das vicilinas, comprovando o resultado obtido com o alinhamento das seq??ncias prim?rias; o motivo R&R, apesar de n?o ter semelhan?a estrutural nas vicilinas, provavelmente tem uma participa??o na atividade de intera??o destas com GlcNac; enquanto que o motivo CD participa diretamente na intera??o das vicilinas com GlcNac. Estes resultados in silico mostram que o n?mero de amino?cidos, os tipos e a quantidade de liga??es feitas pelo motivo CD com GlcNac parecem estar diretamente associados ao poder delet?rio que essas vicilinas possuem para larvas de C. maculatus. Isso pode constutuir um passo inicial na elucida??o de como as vicilinas interagem com quitina in vivo e exercem seu poder t?xico para insetos que possuem membrana peritr?fica

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Herbicides inhibitors of the enzyme acetohydroxyacid synthase (AHAS) present high efficiency in the inhibitory activity with low doses of application and low toxicity for man and the environment. However, several weeds are getting resistence to some classes of herbicides, mainly in the case of AHAS group. Therefore, a proper computational planning of new bioactive compounds is crucial area to model new herbicides. In this study, the enzyme-herbicide interactions were analyzed from the analogous derivated of the pyrimidinylsalicylates group (PSA) which are AHAS inhibitors using quantum- mechanical and molecular docking calculations. The molecular properties obtained after running computer calculation shown that the volume and molecular area can make influence on the inhibition capacity of the ligand, neverthenless, the substituent group has more influence on this parameter. Electronical properties from the HOMO orbitals can certanly make influence on the biological activity due its electron donor capability. The binding free energies of the ligand on the enzyme after docking calculation ranged from - 1.88 to 4.50 kcal mol- 1 , whereas, H, CH3, COCH3 , OH, NO2 and NH2 had the best scored binding energies as substituent groups. Those favorable binding free energies can be justified by the intermolecular interactions between PSAs ligands and AHAS active site residues. In terms of effiency, hydrogen bonds formation can be explained by carboxylate group from the ligands and ARG-377 group from AHAS. / Os herbicidas inibidores da enzima acetohidroxiácido sintase (AHAS) apresentam alta eficiência na atividade inibitória com baixas doses de aplicação com baixa toxicidade para o homem e o meio ambiente. No entanto, várias ervas daninhas estão obtendo resistência a algumas classes de herbicidas, principalmente no caso do grupo AHAS. Portanto, um planejamento computacional adequado de novos compostos bioativos é área crucial para modelar novos herbicidas. Neste estudo, as interações enzima-herbicida foram analisadas a partir do derivado análogo do grupo pirimidinilsalicilato (PSA) que são inibidores de AHAS usando cálculos mecânicoquânticos e de docking molecular. As propriedades moleculares obtidas mostraram que o volume e a área molecular podem influenciar a capacidade de inibição do ligante, mesmo que o grupo substituinte tenha mais influência sobre este parâmetro. As propriedades eletrônicas dos orbitais HOMO podem certamente influenciar a atividade biológica devido à sua capacidade de doação de elétrons. As energias livres de ligação do ligante na enzima após o cálculo de docking variaram de -1,88 a - 4,50 kcal mol- 1 , enquanto que H, CH3, COCH3, OH, NO2 e NH2 apresentaram as melhores energias de ligação pontuadas como grupos substituintes. Estas energias livres de ligação favoráveis podem ser justificadas pelas interações intermoleculares entre ligantes de PSAs e resíduos de sítio ativo de AHAS. Em termos de eficiência, a formação de ligações de hidrogênio pode ser explicada pelo grupo carboxilato partir dos ligantes e do grupo ARG-377 de AHAS. / Dissertação (Mestrado)

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from the Dalbergieae tribe. CTL was isolated by mannose-Sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids and one N-glycosylation site, possessing high similarity with the lectin Platypodium elegans (PELa) and Pterocarpus angolensis (PAL) derived from the same tribe. Two crystal structures of CTL, with monoclinic and tetragonal forms, both complexed with methyl dimanosÃdeo has been solved at 2.25 and 1.9 Ã, respectively, with high similarity. The lectin adopts a typical canonical dimeric organization of legume lectins. The carbohydrate recognition domain (CRD), metal binding site, and glycosylation site have been characterized and the structural basis for interaction with carbohydrate been elucidated. CTL showed acute inflammatory effect in a paw edema model. The protein structure was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, revealing a higher affinity for trimannosides and their interactions were compared with similar lectins, which have the same binding specificity. This is the first report of a crystal structure of a native mannose lectin / specific glucose Dalbergieae tribe with pro-inflammatory activity / Uma lectina glicosilada (CTL) com especificidade a manose e glucose foi detectada e purificada a partir de sementes de Centrolobium Tomentosum, uma leguminosa pertencente à tribo Dalbergieae. CTL foi isolada por cromatografia de afinidade de Sepharose-Manose. A estrutura primÃria foi determinada por espectrometria de massas e consiste em 245 aminoÃcidos e um sitio de ÂNÂ-glicosilaÃÃo, demonstrando similaridade com a lectina de Platypodium elegans (PELa), Pterocarpus angolensis (PAL), dentre outras, oriundas da mesma tribo. Duas estruturas cristalinas de CTL, de formas monoclÃnica e tetragonal, ambas complexadas com metil-dimanosÃdeo, foram resolvidas a 2,25 e 1,9 Ã, respectivamente, apresentando alta similaridade entre si. A lectina mostrou adotar uma organizaÃÃo dimÃrica canÃnica tÃpica de lectinas de leguminosas. O domÃnio de reconhecimento de carboidratos (CRD), local de ligaÃÃo do metal e local de glicosilaÃÃo foram caracterizados e a base estrutural para a interaÃÃo com carboidratos foi elucidado. CTL mostrou efeito inflamatÃrio agudo em um modelo de edema de pata. A estrutura da proteÃna foi submetida a uma anÃlise de interaÃÃes com dimanosÃdeos e trimanosÃdeos por Docking Molecular, revelando sua maior afinidade por trimanosÃdeos e suas interaÃÃes foram comparadas com lectinas similares que possuam a mesma especificidade de ligaÃÃo. Esse à o primeiro relato de estrutura cristalina de uma lectina nativa manose/glucose especÃfica da tribo Dalbergieae com atividade prÃ-inflamatÃria

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-04T13:49:10Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maria Ester Barros_CD_4.pdf: 6351675 bytes, checksum: 1634d753cabb911bd4c0cf8e58182237 (MD5) / Made available in DSpace on 2017-04-04T13:49:10Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Tese Maria Ester Barros_CD_4.pdf: 6351675 bytes, checksum: 1634d753cabb911bd4c0cf8e58182237 (MD5) Previous issue date: 2015-05-01 / CNPQ / A primeira parte deste trabalho envolveu o estudo de docking molecular, a síntese, e a avaliação das atividades antitumorais e anti-inflamatórias de lactonas -insaturadas, análogos estruturais da (-)-Massoialactona, produto natural com conhecida atividade farmacológica. O alvo biológico selecionado para o estudo de docking foi a CRM-1, e tal estudo revelou o potencial biológico dos análogos propostos. Estes análogos foram sintetizados em rendimentos que variaram entre 65-75%, e as suas atividades antitumoral e anti-inflamatória foram avaliadas, sendo que um deles mostrou-se bastante promissor. Estudos posteriores de docking molecular da lactona mais promissora revelou que, apesar dos isômeros R e S assumirem diferentes encaixes no sítio ativo da CRM-1, não há diferença significativa na energia de ligação entre esses isômeros e o alvo molecular estudado. A segunda etapa deste trabalho envolveu o estudo de docking molecular, a síntese, e a avaliação da atividade antitumoral de Z-estilbenos, análogos da Combretastatina A-4 (CA-4). O alvo biológico selecionado para o estudo de docking molecular foi o domínio da colchicina na tubulina. Os cálculos teóricos dos análogos estruturais propostos revelaram que seis desses compostos apresentam diferenças significativas nos valores de energia de ligação quando comparados com o valor teórico calculado para a CA-4, ou seja, teoricamente seriam mais ativos que o produto natural. Dos análogos selecionados a partir do docking molecular, dois foram sintetizados em bons rendimentos empregando-se como precursores teluretos vinílicos e organotrifluoroboratos de potássio. A posterior avaliação da atividade antitumoral dos compostos sintetizados revelou que os resultados obtidos estavam em concordância com o previsto no estudo teórico. / The first part of this work involved the study of molecular docking, synthesis, and the evaluation of anti-tumor and anti-inflammatory activities of unsaturated-lactones, structural analogues of (-)-Massoialactone, a natural product with known pharmacological activity. The biological target selected for the docking study was the CRM-1 and this study revealed the biological potential of the proposed analogues. These analogues were synthesized in yields ranging from 65-75%, and its anti-tumor and anti-inflammatory activities were evaluated, being one of them very promising. Further studies of molecular docking using the most promising lactone revealed that despite the R and S isomers take different fittings in the active site of CRM-1, there is no significant difference in binding energy between these isomers and the molecular target studied. The second step of this work involved the study of molecular docking, synthesis, and evaluation of antitumor activity of Z-stilbenes, analogues of combretastatin A-4. The biological target selected for the study of molecular docking was the domain of colchicine in tubulin. Theoretical calculations of the proposed structural analogues revealed that six of these compounds shown significant differences in binding energy values compared to the theoretical values calculated for the CA-4, ie, theoretically, the proposed compounds would be more active than the natural product. From the selected analogs proposed by molecular docking, two were synthesized in good yields employing vinyl tellurides and potassium organotrifluoroborates as precursors. Further evaluation of the antitumor activity of the synthesized compounds showed that the results were in agreement with the theoretical study.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-11-22T16:54:28Z No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-11-30T15:46:54Z (GMT) No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-30T15:46:54Z (GMT). No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Brazil, where are recorded about 80% of cases worldwide, and has Paracoccidioides sp. as the etiologic agent. Malate synthase (MLS) is an important enzyme related to the fungal metabolism, once it is essential in the glyoxylate cycle, a secondary metabolic pathway of the citric acid cycle exclusive to microorganisms and plants. Its absence in humans makes this enzyme an interesting subject to study, mainly in rational drug design. From recent in vitro studies, several interacting proteins of MLS (receiver) were classified, but the modes of interaction and key regions involved in protein-protein interfaces (PPIs) have not yet been described. In this work, six (6) binding proteins (BPs) were selected to describe the IP's of MLS. Their tridimensonal structures, as well as MLS, were predicted by homology modeling using I-TASSER server, and subsequent molecular dynamics simulations (MD). The most common conformational modes of each protein were obtained by cluster analysis of the trajectories generated by MD. Molecular docking simulations using Gramm-X were then performed for the conformational modes of MLS against the BP's, resulting in a total of 36 complexes. Based on the higher frequency of some small fragments of proteins observed in the IPP's, 57 peptides with sizes between 5 and 20 residues, were initially selected from 5 regions of MLS which are considered more frequent in protein-protein interaction. FlexPepDock simulations were performed to optimize the atomic coordinates of the peptide complexed with MLS, and concomitantly, PepFOLD simulations were performed to evaluate the stability of each peptide in solution. Based on the lower energy score of peptides linked to MLS, and the stability of their structures in solution (MLS-free), 6 peptides were selected as promising ligands to MLS mode 1. The stability and patterns of interactions of these peptides were evaluated in detail. / A paracoccidioidomicose (PCM) é uma micose sistêmica endêmica no Brasil, onde são registrados cerca de 80% dos casos mundiais, e possui como agente etiológico o fungo Paracoccidioides spp. A Malato sintase (MLS) é uma importante enzima relacionada ao metabolismo fúngico, uma vez que é essencial no ciclo do glioxilato, uma via metabólica importante de produção de glicose para parede celular, sendo exclusiva de micro-organismos e plantas. Sua ausência em humanos a torna um alvo interessante de estudo, principalmente, no desenho racional de fármacos. A partir de recentes estudos in vitro, várias proteínas que interagem com a PbMLS (receptor) foram classificadas, porém os modos de interação e as regiões chaves envolvidas nas interfaces proteína-proteína (IPP’s), não foram ainda descritas. Neste trabalho, 6 (seis) proteínas ligantes (PL) foram selecionadas para verificar suas interações com PbMLS. As estruturas tridimensionais dessas proteínas, bem como de PbMLS, foram preditas por homologia, usando o servidor I-TASSER. Simulações de dinâmica molecular (DM) foram realizadas pelo programa GROMACS, e os modos das conformações mais representativas de cada proteína foram determinados baseando-se nas análises de agrupamentos a partir das trajetórias geradas por DM. Simulações de ancoragem molecular com GRAMM-X foram então realizadas entre os modos conformacionais de PbMLS contra os obtidos de PL’s, resultando num total de 36 complexos. Baseado na frequência maior de alguns pequenos fragmentos de proteínas, observados nas IPP’s, 57 peptídeos de tamanhos entre 5 e 20 resíduos de aminoácidos, foram inicialmente selecionados a partir de 5 regiões da PbMLS consideradas mais frequentes na interação proteína-proteína. Simulações com FlexPepDock foram realizadas para otimizar as coordenadas atômicas dos peptídeos complexados com MLS e, concomitantemente, simulações com PepFOLD foram realizadas para avaliar a estabilidade de cada peptídeo em solução. Com base nos mais baixos scores de energia dos peptídeos ligados a MLS bem como na estabilidade de suas estruturas não ligadas em solução , 5 peptídeos foram selecionados como promissores ligantes ao modo 1 de MLS. A estabilidade e os padrões de interações destes peptídeos são avaliados em detalhes.

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.

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&reg; 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&reg; 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.

Le criblage virtuel est de plus en plus utilisé dans les programmes de recherche de nouveaux principes actifs. L’augmentation considérable du nombre de structures résolues a favorisé le recours aux méthodes basées sur la structure de la cible comme le docking. Néanmoins, le choix de la/des structure(s) à utiliser demeure une question d’actualité. Pour tenter d'apporter une réponse, les résultats des études de docking menées sur la banque d’évaluation de référence (DUD) ont été analysés en prenant en compte les propriétés des sites de liaisons des structures de référence. D’intéressants résultats ont été obtenus mettant en évidence l'influence du volume et de l’ouverture des sites actifs sur les performances des méthodes. Ces critères de sélection simples et peu coûteux peuvent servir pour l’optimisation de protocoles de docking.Alors qu’aucune petite molécule inhibitrice du TNFα n’est actuellement commercialisée, l’application d’un protocole hiérarchique de criblage virtuel/in vitro, a permis d’identifier des touches actives. L’une d’elle, de squelette benzènesulfonamide a fait l’objet de pharmacomodulation en vue d’obtenir des analogues optimisés. Vingt molécules inédites ont été synthétisées et testées in vitro et certaines ont montré une activité intéressante. L’ensemble des données obtenues apportent des éléments importants de relation structure-activité. Ces résultats peuvent être exploités pour la conception de molécules innovantes ciblant le TNFα ce qui serait une avancée prometteuse pour le traitement des pathologies liées à une surproduction de cette cytokine comme la polyarthrite rhumatoïde et la maladie de Crohn. / Virtual screening is widely used in drug discovery programs. The increasing number of resolved structures favored the use of Structure Based Virtual Ligand Screening methods like docking. Nevertheless, the choice of the structure(s) used as reference remains a topical issue when several are available. In this work, DUD database docking results were analyzed taking into account the properties of the query structure(s) binding sites. Interesting results were obtained highlighting the influence of active site volume and opening on methods performances. These simple and inexpensive “binding site properties-based” guidelines could be helpful to optimize future docking protocols.Despite important effort, no active small molecule targeting TNFα has been released so far. The use of a virtual/ in vitro hierarchical approach screening allowed identifying some active hits. Starting from one of them with a benzenesulfonamide structure, pharmacomodulation was achieved in order to obtain optimized analogs. Twenty new chemical derivatives with an original structure were synthesized and tested in vitro. Some of them exhibited an interesting activity. Moreover, data obtained provide important elements of structure-activity relationship. These results could constitute the basis for innovative small molecule TNFα-targeted therapeutics which would be a promising step for the treatment of diseases related to overproduction of this cytokine such as rheumatoid arthritis and Crohn's disease.

Pseudomonas aeruginosa est une bactérie à Gram négatif ayant un rôle central dans la morbidité et la mortalité des patients mucoviscidosiques, dont l'environnement pulmonaire particulier favorise les infections chroniques par de nombreux pathogènes opportunistes. Malheureusement, de plus en plus de souches développent des résistances, rendant les antibiothérapies à base de β-lactames de moins en moins efficaces. Parmi les différents mécanismes de défenses développés par P. aeruginosa, l'un des plus important est la détection de l'activité antibiotique, avec en réponse la production de la β-lactamase AmpC, une enzyme qui dégrade les antibiotiques β-lactames. Cette détection met en œuvre la glycosylhydrolase NagZ, qui catalyse la formation de l'inducteur d'AmpC.Récemment au laboratoire, nous avons synthétisé un inhibiteur sélectif de NagZ basé sur une structure azépane. La co-administration à une souche résistante de P. aeruginosa de notre composé et de l'antibiotique β-lactame ceftazidime conduit à une perte de la résistance à l'antibiotique de 50%.Afin d'améliorer la sélectivité et l'activité de notre composé lead, des modifications chimiques du groupement acétamide et du groupement hydroxyle en position C-6 ont été réalisées. L'étude des relations de structure-activité basées sur un cliché cristallographique et sur une étude de docking ont ainsi pu être réalisées. Une autre stratégie explorée a consisté à fonctionnaliser l'atome d'azote endocyclique par un motif sidérophore afin de faciliter la pénétration du composé, ce type de groupement étant en effet connu pour jouer le rôle de cheval de Troie.Les azépanes synthétisés ont été évalués par nos collaborateurs biologistes au Japon et au Canada. / Pseudomonas aeruginosa is a gram negative bacterium playing a major role in morbidity and mortality among CF patients, whose particular pulmonary environment promotes chronic infections by various pathogens1. Unfortunately, more and more bacterial strains are developing resistance, making β-lactam-based antibiotic therapies less effective. Among the different mechanisms of defense developed by P. aeruginosa, one of the most important is the detection of the antibiotic activity by the pathogen, responsively producing the β-lactamase AmpC, an enzyme that degrades the β-lactam antibiotic. This detection implements the glycosylhydrolase NagZ, which catalyzes the formation of the enzyme inducer of AmpC2.We have recently designed a selective inhibitor of NagZ based on an azepane structure. Its co-administration with β-lactam ceftazidime to a β-lactam-resistant strain of P. aeruginosa causes a 50% decrease of the antibiotic resistance3.In order to improve the selectivity and the efficiency of our lead compound, chemical modifications of the acetamide moiety and of the hydroxyl group at C6 have been achieved, allowing to perform a SAR study supported by crystallographic studies and molecular modeling. Another strategy explored has consisted in the functionalization of the endocyclic nitrogen atom of the azepane by a siderophore that will act as a Trojan horse4, in order to improve the penetration of the azepane.The libraries of compounds synthesized were biologically evaluated by our Canadian and Japanese partners.

La thèse porte sur l’étude des 2 premières enzymes de la voie du MEP: la DXS et DXR. La voie du MEP conduit à la biosynthèse des isoprénoïdes chez la plupart des bactéries, dont des pathogènes. Etant absente chez l’homme, les enzymes de cette voie cible idéale pour la recherche de nouveaux antimicrobiens. L’objectif principal était d’améliorer le développement de nouveaux antimicrobiens. Nous avons utilisé des outils computationnels : méthodes de docking et de mécanique moléculaire couplée à la méthode MM/PBSA. Nous avons identifié les résidus contribuant significativement à la fixation d’un ligand dans le site actif de la DXR. Ces résultats ont été utilisés lors de la conception de nouveaux candidats inhibiteurs de type bisubstrat, biligand et difluoro phosphonate, dont 2 ont pu être synthétisés. Nous avons également développé une méthode de synthèse donnant accès au L- et D-GAP énantiomeriquement purs, dans le but d’étudier l’énantiospécificité de la DXS face à son substrat D-GAP. / This thesis concerns the study of the 2 first enzymes of the MEP pathway: DXS and DXR. The MEP pathway permits the biosynthesis of isoprénoïdes in most bacteria, including pathogenic one. As it is not present in human, enzymes of MEP pathway are effective targets in the research of new antimicrobial drugs. The objective was to advance the development of new antimicrobiotic compounds. We used computational tools: molecular docking and molecular dynamics simulations coupled with an MM/PBSA approach. We were able to identify residues that contribute significantly to the ligand binding in the DXR active site. These results were used to guide the conception of new inhibitor models, such as bisubstrates, biligands and α,α-difluoro phosphonates, two of which were synthetized. We also developed a synthesis method to obtain L- and D-GAP as enantiomerically pure molecules. The goal was to study the enantiospecificity of DXS to its substrate, D-GAP.