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

Submitted by PPG Ci?ncia da Computa??o (ppgcc@pucrs.br) on 2017-11-21T17:02:34Z No. of bitstreams: 1 Alan_Diego_dos_Santos_dis.pdf: 1881856 bytes, checksum: cf0113b0b67e0771e4b2920440d41e2b (MD5) / Rejected by Caroline Xavier (caroline.xavier@pucrs.br), reason: Devolvido devido ? falta da folha de rosto (p?gina com as principais informa??es) no arquivo PDF, passando direto da capa para a ficha catalogr?fica. on 2017-11-29T19:03:08Z (GMT) / Submitted by PPG Ci?ncia da Computa??o (ppgcc@pucrs.br) on 2017-11-30T15:50:58Z No. of bitstreams: 1 Alan_Diego_dos_Santos_dis.pdf: 1884320 bytes, checksum: 6e508a972289e66527fd4b76cbae3586 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-12-04T16:14:52Z (GMT) No. of bitstreams: 1 Alan_Diego_dos_Santos_dis.pdf: 1884320 bytes, checksum: 6e508a972289e66527fd4b76cbae3586 (MD5) / Made available in DSpace on 2017-12-04T16:18:35Z (GMT). No. of bitstreams: 1 Alan_Diego_dos_Santos_dis.pdf: 1884320 bytes, checksum: 6e508a972289e66527fd4b76cbae3586 (MD5) Previous issue date: 2017-03-29 / Triagem virtual de bancos de dados de ligantes ? amplamente utilizada nos est?gios iniciais do processo de descoberta de f?rmacos. Abordagens computacionais ?docam? uma pequena mol?cula dentro do s?tio ativo de um estrutura biol?gica alvo e avaliam a afinidade das intera??es entre a mol?cula e a estrutura. Todavia, os custos envolvidos ao aplicar algoritmos de docagem molecular em grandes bancos de ligantes s?o proibitivos, dado a quantidade de recursos computacionais necess?rios para essa execu??o. Nesse contexto, estrat?gias de aprendizagem de m?quina podem ser aplicadas para ranquear ligantes baseadas na afinidade com determinada estrutura biol?gica e, dessa forma, reduzir o n?mero de compostos qu?micos a serem testados. Nesse trabalho, propomos um modelo para ranquear ligantes baseados na arquitetura de redes neurais siamesas. Esse modelo calcula a compatibilidade entre receptor e ligante usando grades de propriedades bioqu?micas. N?s tamb?m mostramos que esse modelo pode aprender a identificar intera??es moleculares importantes entre ligante e receptor. A compatibilidade ? calculada baseada em rela??o ? conforma??o do ligante, independente de sua posi??o e orienta??o em rela??o ao receptor. O modelo proposto foi treinado usando ligantes ativos previamente conhecidos e mol?culas chamarizes (decoys) em um modelo de receptor totalmente flex?vel (Fully Flexible Receptor - FFR) do complexo InhA-NADH da Mycobacterium tuberculosis, encontrando ?timos resultados. / Structure-based virtual screening (SBVS) on compounds databases has been widely applied in early stage of the drug discovery on drug target with known 3D structure. In SBVS, computational approaches usually ?dock? small molecules into binding site of drug target and ?score? their binding affinity. However, the costs involved in applying docking algorithms into huge compounds databases are prohibitive, due to the computational resources required by this operation. In this context,different types of machine learning strategies can be applied to rank ligands, based on binding affinity,and to reduce the number of compounds to be tested. In this work, we propose a deep learning energy-based model using siamese neural networks to rank ligands. This model takes as inputs grids of biochemical properties of ligands and receptors and calculates their compatibility. We show that the model can learn to identify important biochemical interactions between ligands and receptors. Besides, we demonstrate that the compatibility score is computed based only on conformation of small molecule, independent of its position and orientation in relation to the receptor. The proposed model was trained using known ligands and decoys in a Fully Flexible Receptor model of InhA-NADH complex (PDB ID: 1ENY), having achieved outstanding results.

Submitted by Luis Ricardo Andrade da Silva (lrasilva@uefs.br) on 2017-01-11T21:46:05Z No. of bitstreams: 1 Boneco 97-2003-corre??es.pdf: 20164603 bytes, checksum: 78fbb46841fee330fc6bc340e1af3756 (MD5) / Made available in DSpace on 2017-01-11T21:46:06Z (GMT). No. of bitstreams: 1 Boneco 97-2003-corre??es.pdf: 20164603 bytes, checksum: 78fbb46841fee330fc6bc340e1af3756 (MD5) Previous issue date: 2016-08-26 / Funda??o de Amparo ? Pesquisa do Estado da Bahia - FAPEB / Polygalaceae family species are traditionally used in many regions of the world and because of this, research is being conducted to evaluate the biological activities, as well as phytochemicals aspects of these plants. In this way, the present study was to carry out the proposed chemical characterization and evaluation of biological activity in vitro of the extract and prediction of new biological activities in silico of the substances identified in Asemeia ovata (Polyagalaceae). The chemical characterization was made through previous phytochemical screening tests and fingerprint by HPLC-DAD. The isolation and identification of compounds was performed by classical chromatography techniques, HPLC-DAD and 1H and 13C NMR. The evaluation of the antioxidant activity in vitro was taken by Scavenging of DPPH free radical method, acetylcholinesterase activity by adapting the method of Ellman and Artemia salina lethality. The prediction of activity was made by tools for in silico target fishing, followed by docking the DOCK 6.7 program and evaluation of interaction profiles by Protein-Ligand Interaction server profiler. The chemical characterization showed that the extracts are rich in flavonoids and phenolic acids. It was possible to identify and quantify using HPLC-DAD substances: rutin, luteolin-7-O-glucoside, caffeic acid, p-coumaric acid and trans-ferulic acid. Moreover, it was possible to isolate the rutin substance, poligalen and a possible new alkaloid. The ethyl acetate extract was superior in the evaluation of in vitro activity with EC50 = 5.46 mg/mL for antioxidant activity, and LC50 = 71.91 mg/mL A. salina lethality. Acetylcholinesterase activity did not yield significant results (AChEIs% <20%). Tools for in silico target fishing allowed, through the ChemProt 2.0 and DRAR- CPI-servers, to select the molecular targets carbonic anhydrase 12 and epidermal growth factor receptor for routine; for luteolin-7-O-glucoside targets cotransporter 2 sodium / glucose and CDC42-activated protein kinase 1; poligalen to the target protein tyrosine kinase JAK2; and for caffeic acid, p-coumaric acid and trans-ferulic the best targets were epidermal growth factor receptor and Ras-related C3 botulinum toxin substrate 1, carbonic anhydrase 12 and Ornithine carbamoyltransferase, mitochondrial. This work provides new results for the species, both from a chemical and biological point of view, there is good prospects of study with interesting potential to be discovered. / Esp?cies da fam?lia Polygalaceae s?o utilizadas tradicionalmente em muitas regi?es do mundo e, devido a isso, pesquisas est?o sendo realizadas para avaliar as atividades biol?gicas, como tamb?m os aspectos fitoqu?micos desses vegetais. Desta forma, o presente trabalho teve como proposta realizar a caracteriza??o qu?mica e avalia??o de atividades biol?gicas in vitro de extratos da planta inteira e predi??o in silico de novas atividades biol?gicas das subst?ncias identificadas de Asemeia ovata (Polyagalaceae). A caracteriza??o qu?mica foi feita atrav?s de testes de triagem fitoqu?mica pr?via e fingerprint por CLAE-DAD. O isolamento e identifica??o de subst?ncias foi realizado por t?cnicas de cromatografia cl?ssica, CLAE-DAD e RMN de 1H e 13C. A avalia??o da atividade antioxidante in vitro foi feita pelo m?todo de sequestro de radical livre DPPH, atividade anticolinester?sica pela adapta??o do m?todo de Ellman e letalidade frente Artemia salina. A predi??o de atividades in silico foi feita por m?todos de Triagem Virtual Inversa (TVI), seguido de reacoplamento pelo programa DOCK 6.7 e avalia??o dos perfis de intera??o pelo servidor Protein-Ligand Interaction Profiler. A caracteriza??o qu?mica mostrou que os extratos s?o ricos em ?cidos fen?licos e flavonoides. Foi poss?vel identificar e quantificar, atrav?s de CLAE-DAD, as subst?ncias: rutina, luteolina-7-O-glicos?deo, ?cido cafeico, ?cido p-cum?rico e ?cido trans-fer?lico. Al?m disso, foi poss?vel isolar as subst?ncias rutina, poligaleno e um poss?vel novo alcaloide. O extrato acetato de etila mostrou-se superior na avali??o das atividades in vitro, com CE50 = 5,46 mg/mL para atividade antioxidante, e CL50 = 71,91 ?g/mL para letalidade frente a A. salina. Para a atividade anticolinester?sica n?o obteve-se resultados significativos (%IAChE < 20%). A TVI permitiu selecionar, atrav?s dos servidores ChemProt 2.0 e DRAR-CPI, os alvos moleculares Anidrase carb?nica 12 e Receptor de fator de crescimento epid?rmico para a rutina; para a luteolina-7-O-glicos?deo os alvos Cotransportador 2 de s?dio/glicose e Prote?na quinase CDC42 ativada 1; para o poligaleno o alvo Prote?na tirosina quinase JAK2; e para os ?cidos cafeico, p-cum?rico e trans-fer?lico os melhores alvos foram Receptor de fator de crescimento epid?rmico e Ras-relacionada ao substrato C3 da Toxina botul?nica 1, Anidrase carb?nica 12 e Ornitina carbamoiltransferase, mitocondrial. Esse trabalho fornece resultados in?ditos para a esp?cie, tanto do ponto de vista qu?mico, como biol?gico, apresentando boas perspectivas de estudo, com interessante potencial a ser descoberto.

隨著個人計算機運算能力的快速發展，虛擬藥物篩檢已被廣泛運用。目前運用於計算機輔助藥物虛擬篩選的化合物數據庫多為人工合成的數據庫，而用於天然產物藥物篩選的數據庫則較少報道。為了加速天然化合物的虛擬篩選，我們建立了包含約8000個天然產物的數據庫。他們中的大多數為傳統中藥。 / 為了驗證天然產物數據庫的可用性，其被用於篩選乙酰膽鹼酯酶抑製劑。該數據庫成功地確定了美國藥品監督管理局所批準的乙酰膽鹼酯酶抑製劑，如石杉鹼甲和他克林，表示該天然產物數據庫可以用於藥物虛擬對接篩選。 / 除了已知的乙酰膽鹼酯酶抑製劑，十二種植物化學物（大黃酸，大黃素，蘆薈大黃素，大黃酚，花椒毒素，珊瑚菜素，別異歐前胡素，歐前胡素，紫草素，乙酰紫草素，異戊紫草素和β，β-二甲基丙烯酰紫草素）被確定為新的乙酰膽鹼酯酶抑製劑。澱粉樣蛋白聚集可以導致神經細胞死亡；本研究中新發現的乙酰膽鹼酯酶抑製劑乙酰紫草素能夠阻止澱粉樣蛋白的聚集。除此之外，乙酰紫草素及其衍生物可以對抗過氧化氫誘導的神經細胞凋亡。其抗凋亡的活性作用是通過抑制活性氧的產生，以及保護線粒體膜電位的損失所實現的。亞鐵血紅素加氧酶在其神經細胞保護作用中起重要作用。 / 趨化因子受體4為跨膜G蛋白偶聯受體（GPCRs）。 CXCR4已被確定為一個新治療以及預防腫瘤轉移的新靶點。本研究利用分子對接篩選，從天然產物數據庫篩選選出CXCR4拮抗劑。通過分子對接和基於細胞的測定，黃芪甲苷，羥基紅花黃色素A和水飛薊賓已被確定為CXCR4拮抗劑。抗轉移的研究表明，黃芪甲苷和水飛薊賓抑制CXCL12誘導乳腺癌細胞的遷移和侵襲。此外，水飛薊賓也抑制CXCL12誘導的人臍靜脈內皮細胞管形成。另一方面，羥基紅花黃色素A對乳腺癌細胞的增殖表現出較強的抑製作用，因此很難進行抗轉移實驗。 / With the rapid advances in personal computing power, virtual drug screening has become increasingly popular. While there are numerous databases for synthetic compounds, there are few natural product databases that are specifically for in silico docking studies. To facilitate virtual docking on natural compounds, in-house Natural Products Database has been established, which contains approximately 8,000 naturally occurring chemicals so far. Most of them are documented Traditional Chinese Medicines. / In order to validate the usefulness of the database, in silico screening of acetylcholinesterase inhibitors (AChEIs) by virtual docking was performed. The database successfully identified the FDA-approved AChEIs such as huperzine and tacrine, indicating the in-house database is workable for natural products docking screening. / Apart from well-known AChE inhibitors, twelve phytochemicals (emodin, aloe-emodin, chrysophanol, rhein, xanthotoxin, phellopterin, alloisoimperatorin, imperatorin, shikonin, acetylshikonin, isovalerylshikonin and β, β-dimethylacrylshikonin) were identified as AChE inhibitors in this study that were not previously reported. Amyloid aggregation leads to toxic species that cause neuronal cell deaths, it was found that the newly identified AChEIs acetylshikonin and shikonin are able to prevent amyloid aggregation. A series of cell-based analysis were conducted for in vitro evaluation of the neuroprotective activities of the newly identified AChEIs. Acetylshikonin and its derivatives was found to prevent apoptotic cell death induced by hydrogen peroxide in human and rat neuronal SH-SY5Y and PC12 cells at 10 μM. Acetylshikonin exhibited the most potent anti-apoptotic activity through inhibition of reactive oxygen species (ROS) generation as well as protection of the loss of mitochondria membrane potential. Furthermore, acetylshikonin upregulates hemooxygenase 1(HO-1) which is a key step mediating its anti-apoptotic activity from oxidative stress in SH-SY5Y cells. / The C-X-C chemokine receptor type 4 (CXCR4) belongs to the class A family of seven transmembrane G protein-coupled receptors (GPCRs). CXCR4 has been identified as one of novel target against metastasis. A search for natural CXCR4 antagonists was conducted from natural product database by molecular docking for anti-metastasis study. Astragaloside IV, hydroxy safflower yellow A and silibinin have been identified as novel CXCR4 antagonists by both molecular docking and characterized by various cell-based assays. Anti-metastasis study showed that astragaloside IV and silibinin inhibited CXCL12-induced migration and invasion in breast cancer cells. In addition, silibinin also inhibited CXCL12-induced tube formation in human umbilical vein endothelial cells. On the other hand, hydroxy safflower yellow A exhibited a strong cytotoxicity on breast cancer cell proliferation, which is difficult to conduct anti-metastasis experiments. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wang, Yan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 230-250). / Abstracts also in Chinese.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-14T17:55:12Z No. of bitstreams: 1 RosaHelenaSilva.pdf: 2716670 bytes, checksum: 07dee1e77d704e91281c33c31e8c4938 (MD5) / Made available in DSpace on 2017-06-14T17:55:12Z (GMT). No. of bitstreams: 1 RosaHelenaSilva.pdf: 2716670 bytes, checksum: 07dee1e77d704e91281c33c31e8c4938 (MD5) Previous issue date: 2016-10-19 / Borreria verticillata (L.) G. Mey species known as broom vassourinha has antibacterial, antimalarial, hepatoprotective, antioxidative, analgesic and antiinflammatory activities; however, its antinociceptive action still demands more thorough investigation. The present study was to assess the antinociceptive activity of B. verticillata crude hydroalcoholic extract (EHBv) and the ethyl acetate fraction (FAc) by means of in vivo and in silico studies. In vivo assessment included the paw edema test, the writhing test, the formalin test and the tail flick test. Wistar rats and Swiss mice were divided into 6 groups and given the following treatments oral: 0.9% NaCl control group (CTL), 10 mg/kg memantine (MEM), 10 mg/kg indomethacin (INDO), 500 mg/kg EHBv (EHBv 500), 25 mg/kg FAC (FAc 25), 50 mg/kg and FAc (FAC 50). EHBv, FAc 25 and 50 treatments exhibited anti-edematous and peripheral antinociceptive effects. For in silico assessment, compounds found in FAc were subjected to molecular docking, and the leading compound was selected for molecular dynamics (MD) simulations. Ursolic acid exhibited better affinity parameters with the enzyme COX-2 and the NMDA receptor subunits GluN1a and GluN2B on molecular docking. In MD simulations, AU exhibited highly frequent interactions with residues Arg120 and Glu524 in the COX-2 active site and NMDA, whereby it might prevent COX-2 and NMDA receptor activation. Treatment with ursolic acid 10mg / Kg (AU) showed peripheral and central antinoceceptivo effect. The antinociceptive effect of B. verticillata might be predominantly attributed to peripheral actions, including the participation of anti-inflammatory components. Ursolic acid is the main active component and seems to be a promising source of COX-2 inhibitors and NMDA receptor antagonists / Borreria verticillata (L.) G. Mey espécie conhecida como vassourinha apresenta atividade antibacteriana, antimalárica, hepatoprotetora, antioxidante, analgésica e anti-inflamatória, entretanto sua atividade antinociceptiva é pouco estudada. O objetivo deste trabalho foi avaliar atividade antinociceptiva do extrato hidroalcoólico bruto (EHBv) e fração acetato de etila (FAc) de B. verticillata realizando estudos in vivo e in silico. Para avaliação in vivo, foram utilizados os testes do edema de pata, contorções abdominais, formalina e tail flick. Ratos Wistar e camundongos Swiss foram tratados via oral e divididos em 6 grupos: controle-NaCl 0.9%(CTL), memantina 10 mg/Kg (MEM), indometacina 10 mg/Kg (INDO), EHBv 500 mg/kg (EHBv 500), FAc 25 mg/Kg (FAc 25), FAc 50 mg/Kg (FAc 50). O tratamento com EHBv 500, FAc 25 e 50 apresentou efeito antiedematogênico e antinociceptivo periférico. Para avaliação in silico os compostos identificados na FAc foram submetidos a docagem molecular, o melhor composto foi selecionado para simulações de dinâmica e testado in vivo molecular. O ácido ursólico apresentou melhores parâmetros de afinidade com COX-2, GluN1a e GluN2B durante a docagem molecular. Nas simulações por dinâmica molecular, o ácido ursólico apresentou alta frequência de contatos com Arg120 e Glu524 do local ativo da COX- 2 e com o domínio LBD da Glun1a e GluN2B podendo com isso, impedir a ativação da COX-2 e do receptor NMDA. O tratamento com ácido ursólico 10mg/Kg (AU) apresentou efeito antinoceceptivo periférico e central. Sugere-se que o efeito antinociceptivo periférico de B. verticillata pode ser atribuído predominantemente à ação de compostos com ação anti-inflamatória. O ácido ursólico é o principal composto ativo, sendo um composto promissor para o desenvolvimento de fármacos inibidores da COX-2 e antagonistas dos receptores NMDA.

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.

Carbohydrates are abundant in nature and have functions ranging from energy storage to acting as structural components. Analysis of carbohydrate structures is important and can be used for, for instance, clinical diagnosis of diseases as well as in bacterial studies. The complexity of glycans makes it difficult to determine their structures. NMR spectroscopy is an advanced method that can be used to examine carbohydrates at the atomic level, but full assignments of the signals require much work. Reliable automation of this process would be of great help. Herein studies of Escherichia coli O-antigen polysaccharides are presented, both a structure determination by NMR and also research on glycosyltransferases which assemble the polysaccharides. The computer program CASPER has been improved to assist in carbohydrate studies and in the long run make it possible to automatically determine structures based only on NMR data. Detailed computer studies of glycans can shed light on their interactions with proteins and help find inhibitors to prevent unwanted binding. The WaaG glycosyltransferase is important for the formation of E. coli lipopolysaccharides. Molecular docking analyses of structures confirmed to bind this enzyme have provided information on how inhibitors could be composed. Noroviruses cause gastroenteritis, such as the winter vomiting disease, after binding human histo-blood group antigens. In one of the projects, fragment-based docking, followed by molecular dynamics simulations and binding free energy calculations, was used to find competitive binders to the P domain of the capsid of the norovirus VA387. These novel structures have high affinity and are a very good starting point for developing drugs against noroviruses. The protein targets in these two projects are carbohydrate binding, but the techniques are general and can be applied to other research projects. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Submitted. Paper 5: Manuscript. Paper 6. Manuscript.

L’ADN, support de l’information génétique, est constamment altéré par des agents physiques ou chimiques d’origines endogènes (métabolisme) et exogènes (UV, radiations ionisantes, produits chimiques) dont les effets sont génotoxiques. Ces modifications structurales délétères de l’ADN sont éliminées par de nombreux mécanismes de réparation. Parmi eux, le système de réparation par excision de bases (BER) est initié par les ADN glycosylases qui reconnaissent et éliminent les bases endommagées. Dans certaines stratégies anti-cancéreuses, l’utilisation de la chimiothérapie et la radiothérapie ont pour but la destruction des cellules cancéreuses en altérant leur ADN. Dans ce contexte, les ADN glycosylases réparent l’ADN des cellules traitées et induisent une résistance non désirée au traitement, faisant de ces enzymes des cibles thérapeutiques intéressantes. Le but de ces travaux est d’approfondir la compréhension des mécanismes de réparation des ADN glycosylases de la superfamille structurale Fpg/Nei grâce à la modélisation moléculaire et de pouvoir identifier et concevoir des inhibiteurs de ces enzymes. Les simulations de dynamique moléculaire (DM) nous ont permis d’étudier la « Lesion Capping Loop » (LCL) et de l’associer à la stabilisation de la base endommagée positionnée dans le site actif. Nous avons également étudié les chemins de sortie possibles de la base après coupure par l’enzyme et l’implication de la boucle LCL dans ce phénomène grâce à des simulations de DM ciblée (TMD-1). De plus, les simulations de DM couplées à un protocole d’amarrage moléculaire « aveugle » nous ont permis d’identifier 2 sites de fixations possibles majoritaires pour des petites molécules potentiellement inhibitrices. Un de ces sites correspondant au site actif de hNEIL1 a fait l’objet d’un criblage virtuel d’une partie de la base de molécules Ambinter. Ceci nous a permis d’identifier des molécules potentiellement inhibitrices dont les effets seront prochainement testés in vitro dans l’équipe sur la protéine humaine hNeil1. / The DNA, genetic information support, is frequently damaged by physical or chemical agents from endogenous (cell metabolism) and exogenous (UV, ionizing radiations, chemicals) factors whose effects are genotoxic. These deleterious DNA structural alterations are removed by many DNA repair mechanisms. Among them, the base excision repair (BER) is initiated by DNA glycosylases which recognize and remove damaged bases. In some anti-cancer strategies, the use of chemo- and radiotherapy is aimed to cancerous cells destruction by altering their DNA. In that specific context, DNA glycosylases repair the DNA of treated cells and induce unwanted resistance to treatments, making these enzymes interesting therapeutic targets. The purpose of this work is to deepen the repair mechanism knowledge of Fpg/Nei structural superfamily of DNA glycosylases using molecular modeling and designing inhibitors of these enzymes. Molecular dynamic simulations allowed us to study the « Lesion Capping Loop » (LCL) and to associate its role to substrate stabilization in the enzyme active site. We also studied some possible excision’s product release pathways and LCL implication in this phenomena by targeted molecular dynamic simulations (TMD-1). Furthermore, molecular dynamic simulations coupled to a blind molecular docking protocol allowed us to identify 2 possible main binding sites of potential inhibitiors. One of these binding sites corresponding to the hNEIL1 active site has been the object of a virtual screening of the Greenpharma database. This allowed us to identify potential inhibitors whom effects will be soon tested in vitro on the humain protein hNEIL1.

The development of novel imidazopyridines, which includes both tetrahydroimidazo[1,5-a]pyridine (rIMP) and imidazo[1,5-a]pyridine (IMP) was investigated using conventional and microwave induced procedures that afforded compounds at high yield of 88-96%. rIMP was synthesized using a two-step procedure that involved the microwave synthesis of IMP, then the reduction of the pyridine moiety of the fused imidazopyridine rings using 10% Pd/C and hydrazine monohydrate. The microwave synthesis of imidazopyridines involved the one pot reaction of 2-benzoylpyridine, substituted benzaldehyde and ammonium formate in acetic acid under open vessel microwave conditions, which resulted in products within 40 minutes. Novel PEG-IMP development, involved the synthesis of ethylene glycol tethered benzaldehydes and IMPs using traditional Williamson etherification synthesis, which afforded products at a high yield of 92-95%. We have then shown IMP and rIMP roles in its antiproliferative property towards PCa cells, specificity in inhibiting PI3K isoforms, and structural motif’s interaction with different residues in the kinase binding domain of the class I PI3K isoforms. The antiproliferative property towards PC3 cells shows increased activity with compounds containing pyridyl group on carbon 3 of the imidazo[1,5-a]pyridine parent moiety with signs of toxicity to PC3 within 24 hours of incubation and at 1 μM of the parent compound. Furthermore, the IMPs were tested against five prostate cellular lines: PC3, RWPE1, D145, LNCaP and LNCaP C81. IMPs showed little activity towards RWPE1 and increased activity towards PC3 cells. We determined that functionalizing the phenyl group at position 1 increased the efficacy of rIMP compared to the IMP. After showing increased toxicity to PC3 cells, it was important to investigate the mechanism in which IMP pose toxicity towards PC3 cells. The biochemical assay showed that rIMP was more effective in inhibiting PI3Kα isoform compared to both pan inhibitor wortmannin and IMP. Both IMP and rIMP inhibited more than 60% of PI3Kγ isoform activity at nanomolar concentrations. After showing IMPs affinity to PI3K isoforms, we investigated the binding interactions rIMP and IMP towards the PI3K isoforms using MOE molecular modeling software.

La drépanocytose est une maladie génétique qui se caractérise par des globules rouges en forme de faucille. Chez les personnes atteintes de drépanocytose, ces globules rouges (GR) adhèrent à l’endothélium vasculaire et provoquent ainsi une vaso-occlusion. Ce phénomène s’explique par la surexpression de la protéine Lutheran (Lu) à la surface des globules rouges falciformes qui se lie fortement à la Laminine (Ln) 511/521 exprimée par l’endothélium vasculaire enflammé. Le but de cette étude est d’identifier un inhibiteur d’interaction protéine-protéine (PPI) qui possède une forte probabilité de liaison à Lu afin d’inhiber l’interaction Lu-Ln 511/521. Un criblage virtuel de 1 295 678 composés ciblant la protéine Lu a été réalisé. La validation préalable d’un protocole de scoring a été envisagée sur la protéine CD80 qui présente un site de liaison avec des caractéristiquestopologiques et physico-chimiques similaires au site de liaison prédit sur Lu ainsi que plusieurs ligands avec des constantes d’affinité connues. Ce protocole contient différentes étapes de sélection basées sur les affinités calculées (scores), des simulations de dynamique moléculaire et les propriétés moléculaires. Un protocole de scoring fiable a été validé sur CD80 avec le programme de docking DOCK6 et les fonctions de scoring XSCORE et MM-PBSA ainsi qu’avec la méthode decalcul FMO. L’application de ce protocole sur Lu a permis d’obtenir deux ligands validés par des tests in vitro qui font l’objet d’un dépôt de brevet. La fonction de scoring XSCORE a permis d’identifier neuf autres ligands qui semblent aussi être des candidats prometteurs pour inhiber l’interaction Lu-Ln 511/521. / Drepanocytosis is a genetic blood disorder characterized by red blood cells that assume an abnormal sickle shape. In the pathogenesis of vaso-occlusive crises of sickle cell disease, red blood cells bind to the vascular endothelium and promote vaso-occlusion. At the surface of these sickle red blood cells, the overexpressed protein Lutheran (Lu) strongly interacts with the Laminin (Ln) 511/521.The aim of this study was to identify a protein-protein interaction (PPI) inhibitor with a highprobability of binding to Lu for the inhibition of the Lu-Ln 511/521 interaction. A virtual screening was performed with 1 295 678 compounds that target Lu. Prior validation of a robust scoring protocol was considered on the protein CD80 because this protein has a binding site with similar topological and physico-chemical characteristics and it also has a series of ligands with known affinity constants. This protocol consisted of multiple filtering steps based on calculated affinities (scores), molecular dynamics simulations and molecular properties. A robust scoring protocol was validated on the protein CD80 with the docking program DOCK6 and the scoring functions XSCORE and MM-PBSA and also with the FMO method. This protocol was applied to the protein Lu and we found two compounds that were validated by in vitro studies. The protection of these ligands by a patent is under process. Nine other compounds were identified by the scoring functionXSCORE and seem to be promising candidates for inhibiting the Lu-Ln 511/521 interaction.