Même si le docking protéine-protéine devient un outil incontournable pour répondre aux problématiques biologiques actuelles, il reste cependant deux difficultés inhérentes aux methodes actuelles: 1) la majorité de ces méthodes ne considère pas les possibles déformations internes des protéines durant leur association. 2) Il n'est pas toujours simple de traduire les informations issues de la littérature ou d'expérimentations en contraintes intégrables aux programmes de docking. Nous avons donc tenté de développer une approche permettant d'améliorer les programmes de docking existants. Pour cela nous nous sommes inspirés des méthodologies mises en place sur des cas concrets traités durant cette thèse. D'abord, à travers la création du complexe ERBIN PDZ/Smad3 MH2, nous avons pu tester l'utilité de la Dynamique Moléculaire en Solvant Explicite (DMSE) pour mettre en évidence des résidus importants pour l'interaction. Puis, nous avons étendu cette recherche en utilisant divers serveurs de docking puis la DMSE pour cibler un résultat consensus. Enfin, nous avons essayé le raffinage par DMSE sur une cible du challenge CAPRI et comparé les résultats avec des simulations courtes de Monte-Carlo. La dernière partie de cette thèse portait sur le développement d'un nouvel outil de visualisation de la surface moléculaire. Ce programme, nommé MetaMol, permet de visualiser un nouveau type de surface moléculaire: la Skin Surface Moléculaire. La distribution des calculs à la fois sur le processeur de l'ordinateur (CPU) et sur ceux de la carte graphique (GPU) entraine une diminution des temps de calcul autorisant la visualisation, en temps réel, des déformations de la surface moléculaire. / Protein-protein docking has become an extremely important challenge in biology, however, there remain two inherent difficulties: 1) most docking methods do not consider possible internal deformations of the proteins during their association; 2) it is not always easy to translate information from the literature or from experiments into constraints suitable for use in protein docking algorithms. Following these conclusions, we have developed an approach to improve existing docking programs. Firstly, through modelling the ERBIN PDZ / Smad3 MH2 complex, we have tested the utility of Molecular Dynamics with Explicit Solvent (MDSE) for elucidating the key residues in an interaction. We then extended this research by using several docking servers and the DMSE simulations to obtain a consensus result. Finally, we have explored the use of DMSE refinement on one of the targets from the CAPRI experiment and we have compared those results with those from short Monte-Carlo simulations. Another aspect of this thesis concerns the development of a novel molecular surface visualisation tool. This program, named MetaMol, allows the visualisation of a new type of molecular surface: the Molecular Skin Surface. Distributing the surface calculation between a computer's central processing unit (CPU) and its graphics card (GPU) allows deformations of the molecular surface to be calculated and visualised in real time.

Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...

Dans la recherche de nouveaux traitements des maladies broncho-pulmonaires comme l'asthme et la broncho-pneumopathie chronique obstructive, les inhibiteurs de PDE4 sont des cibles intéressantes. Dans cette voie, notre laboratoire s'intéresse à la synthèse d'une nouvelle famille d'inhibiteurs à base pyridazinone. Pour cela, cette thèse couple la modélisation moléculaire (docking : développement méthodologique et application), la synthèse organique ainsi que des tests biologiques.Dans un premier temps, le développement du logiciel d'amarrage moléculaire AlgoGen a été poursuivi. AlgoGen (créé initialement à l'université de Lorraine en 2009) est un logiciel qui couple un algorithme génétique pour la recherche conformationnelle à une évaluation de l'énergie protéine-ligand à un niveau quantique semi-empirique alors que les autres logiciels existant effectuent ce calcul à un niveau classique en général. Le calcul d'une énergie à un niveau quantique est très coûteux en temps. C'est pourquoi, nous avons apporté de nombreuses modifications à ce logiciel afin d'accroître son efficacité dans la recherche conformationnelle. Ce logiciel a ensuite été utilisé sur un jeu de 22 dimères (typiques des reconnaissances moléculaires biologiques) et à huit systèmes protéine-ligand.Dans un deuxième temps, huit inhibiteurs ont été synthétisés et testés in vitro sur la cible PDE4. Pour compléter ce volet expérimental, une étude de structure-activité a été effectuée grâce au docking moléculaire (AlgoGen, Autodock, Glide) pour rationaliser les activités mesurées (IC50). Pour terminer, des pharmaco-modulations guidées par docking ont été réalisées afin de proposer de nouveaux inhibiteurs de plus grandes affinités avec la protéine PDE4D. / For the research of new treatment of bronchopulmonary diseases such as asthma and chronic obstructive pulmonary disease (COPD), the PDE4 inhibitors are an attractive target. Our laboratory is interested in a new PDE4 inhibitors family based on the pyridazinone pattern. For this purpose, this thesis couples molecular modeling (docking: methodological development and application), organic synthesis and biological tests.First, the development of the molecular docking software AlgoGen was continued. AlgoGen (initially created at university of Lorraine in 2009) is a program which couples a genetic algorithm for the conformational research and a protein-ligand energy evaluation at the quantum semi-empirical level while other software do this evaluation at a classical level. Quantum energy calculations are very time consuming. That is the reason why some modifications have been made to improve its efficiency for the conformational search. This software was then used for calculations on a set of 22 dimers (typical in biological molecular recognition) as well on 8 ligand-protein complexes.Secondly, eight inhibitors were synthesized and tested in vitro on the PDE4 target. To complete the experimental part, a structure-activity relationship study was carried out through a molecular docking to rationalize the measured activity (IC50). Finally, pharmaco-modulations guided by docking were made to propose new inhibitors with more affinity with the protein.

As estruturas e propriedades biológicas das amidas piplartina e a piperina, isoladas respectivamente de Piper tuberculatum e P. nigrum, inspiraram a síntese de 89 derivados e 7 esters estruturalmente relacionadas. As preparações envolveram metodologias tradicionais e os compostos purificados tiveram suas estruturas caracterizadas por análises espectroscópicas e espectrométricas. Os estudos de fragmentação por IE e IES indicaram a clivagem preferencial da ligação N-CO no caso das cinamamidas, dienamidas e cinamimidas. Estudos computacionais envolvendo afinidade protônica e energias de ligação confirmaram a fragmentação preferencial da ligação amídica para as amidas. A citotoxicidade de 89 substâncias foi avaliada contra três células leucêmicas (K562, Nalm6 e Raji) e a partir dos valores de IC50 foram realizados estudos de relação estrutura-atividade (SAR). As linhagens K562 e a Nalm6 foram a mais resistente e vulnerável, respectivamente, e as amidas piplartina (1a), N-Ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4,5-trimetoxifenil)propanamida (1n), e (E)-N,N-dibutil-3-(3,4-dimetoxifenil)acrilamida (13h) foram as mais ativas com IC50 de 0,34 µM; 0,84 µM e 1,88 µM contra K562 e (E)-N-ciclohexil-N-(ciclohexilcarbamoil)-3-(3,4-dimetoxifenil)acrylamida (13i) com IC50 de 0,98 µM contra Nalm6. A avaliação de atividade leishmanicida de 18 substâncias não se mostrou promissora. As abordagens qualitativas e quantitativas foram feitas baseadas nos descritores moleculares gerados pelo programa VolSurf+. A partir de métodos quimiométricos tais com PLS, algoritmo genético, árvores de decisão foi possível gerar modelos para correlacionar às propriedades moleculares com a atividade biológica. As propriedades de absorção, distribuição, metabolismo e excreção e os equilíbrios entres as regiões hidrofílicas e hidrofóbicas foram importantes para atividade citotóxica. O estudo de ancoragem molecular mostrou que as amidas (E)-N,N-dibutil-3-(3,4,5-trimetoxifenil)acrilamida (1l), 1n, (E)-3-(4-clorofenil)-N-ciclohexil-N-(ciclohexilcarbamoil)acrilamida (5a), 13h e 13i podem atuar como inibidores das histonas desacetilases particularmente HDAC4 e HDAC8. / The structures and biological properties of the amides piplartine and piperine isolated from Piper tuberculatum and P. nigrum respectively, inspired the synthesis of derivatives 89 and 7 esters structurally related. Their preparations were achieved using classical procedures and the purified amides were submitted to spectroscopic and spectrometric characterization. The study of fragmentation process by EI and ESI suggested the preferential cleavage of the N-CO bond of cinnamamides, dienamides and cinnamimides. The cytotoxicity of 89 compounds was evaluated against three leukemic cells (K562, Nalm6 and Raji) and based on IC50 values the structure-activity relationship (SAR) was performed. While the K562 and Nalm6 cells were the more resistant and more sensitive, respectively, the amides piplartine (1a), N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4,5-trimethoxyphenyl)propanamide (1n) and (E)-N,N-dibutyl-3-(3,4-dimethoxyphenyl)acrylamide (13h) were in general the most active with IC50 of 0.34 µM, 0.84 µM and 1.88 µM against K562 and (E)-N-cyclohexyl-N-(ciclohexylcarbamoyl)-3-(3,4-dimethoxyphenyl)acrylamide (13i) with IC50 of 0.98 µM against Nalm6. The evaluation of leishmanicidal activity of 18 substances was also performed but was not promising. Qualitative and quantitative approaches were made based on molecular descriptors generated by VolSurf+ program. The chemometric methods such as PLS, genetic algorithm, decision trees generated models to correlate molecular properties with the biological activity. The absorption, distribution, metabolism and excretion properties and a balance between hydrophilic and hydrophobic moieties of the amides were important for an optimized activity. The molecular docking revealed that amides such as (E)-N,N-dibutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (1l), 1n, (E)-3-(4-chlorophenyl)-N-cyclohexil-N-(cyclohexylcarbamoyl)acrylamide (5a), 13h and 13i have potential to act as possible inhibitors of histone deacetylase proteins particularly HDAC4 and HDAC8.

As metaloproteinases de matriz (MMP) são enzimas superexpressas em quase todos os tumores humanos, sendo que os subtipos MMP-2 e MMP-9 têm sido associados ao potencial metastático e prognóstico desfavorável em neoplasias malignas como, por exemplo, melanoma metastático e glioma. Compostos capazes de inibir a atividade destas enzimas podem representar potenciais agentes terapêuticos. O composto 4-nerolidilcatecol (4-NC), isolado de plantas do gênero Pothomorphe, apresentou resultados promissores para o tratamento do melanoma e glioma e foi capaz de atuar em várias etapas bioquímicas importantes envolvidas na progressão dessas patologias, inclusive inibindo MMP-2 e MMP-9. No entanto, o mecanismo de ação do 4-NC não está completamente elucidado. O presente estudo envolveu a aplicação de métodos de modelagem molecular e de formalismos do planejamento de novas moléculas auxiliado por computador, CAMD (Computer-Aided Molecular Design) a fim de explorar a interação entre esta molécula e as enzimas MMP-2 e MMP-9, além de planejar novos inibidores para estes alvos. Análise exploratória de dados, que compreende a análise de agrupamentos hierárquicos e de componentes principais. foi desenvolvida para um conjunto de hidroxamatos (N=64) descritos como inibidores de MMP-2 e MMP-9, a fim de identificar as propriedades moleculares que mais influenciavam o processo de discriminação dos compostos. As propriedades termodinâmicas, eletrônicas e estéricas foram importantes para descrever os compostos mais ativos no conjunto de dados da MMP-2. Para a MMP-9, o coeficiente de distribuição (ClogD) em pH 1,5 foi relevante no processo de discriminação do conjunto. A presença de substituintes volumosos na porção R3 parece ser crucial para o conjunto de inibidores investigados. Esta região está envolvida em interações moleculares com a cavidade S1 de ambas as enzimas, mas há um limite de volume a ser considerado para estes substituintes. O formalismo QSAR-4D independente do receptor (IR) foi aplicado ao mesmo conjunto de dados e permitiu estabelecer o mapeamento do farmacóforo, além de explorar diferentes alinhamentos para a obtenção da hipótese de conformação bioativa prevista pelo melhor modelo de QSAR. OS modelos QSAR apresentaram boa capacidade de previsão, auxiliaram na proposição de novos inibidores e estimaram a atividade do 4-NC. Com o melhor modelo QSAR para MMP-9 (N=64), a atividade prevista para o 4-NC foi classificada na faixa dos inibidores com atividade moderada. Entretanto, o melhor modelo QSAR obtido para MMP-2 (N=38) não foi capaz de prever, de forma adequada, a atividade de compostos com arcabouço químico diferente daqueles utilizados na construção dos modelos. Estudos de ancoramento molecular foram desenvolvidos para investigar a orientação do 4-NC no sitio catalítico das duas enzimas e as interações que poderiam ser estabelecidas nestes complexos. Duas conformações favoráveis foram encontradas. Simulações computacionais de dinâmica molecular foram desenvolvidas com os complexos mais promissores selecionados nos estudos de ancoramento, a fim de obter informações mais detalhadas e de maior confiabilidade. sobre suas interações intermoleculares. O 4-NC tende a se orientar no sítio de forma a acomodar sua cadeia lateral no bolso S1 adjacente ao sítio catalítico em ambas as enzimas. Ensaios de zimografia também foram realizados com o objetivo de elucidar possíveis contribuições da cadeia lateral e do núcleo catecólico do 4-NC na atividade inibitória frente às enzimas em estudo. O núcleo catecólico parece ser o responsável por sua atividade, pois o composto 1,2dimetoxibenzeno, que possui as hidroxilas bloqueadas por grupos metil, não foi capaz de exercer atividade inibitória significante frente à MMP-2 e MMP-9. Estudos de voltametria reforçaram a hipótese de que o 4-NC tem a capacidade de quelar os íons zinco presentes no tampão de incubação. / Matrix metalloproteinases (MMP) enzymes are overexpressed in almost all human tumors, and MMP-2 and MMP-9 subtypes have been associated with metastatic potential and poor prognosis in malignant tumors, such as metastatic melanoma and glioma. Compounds capable of inhibiting the activity of theses enzymes would be considered as potential therapeutic agents. The 4-nerolidylcatechol compound (4-NC), isolated from plants of genus Pothomorphe, has showed promising results in the treatment of melanoma and glioma, and was able to act in several important biochemical steps involved in the progression of these diseases, as well as inhibiting MMP-2 and MMP-9. However, the 4-NC mechanism of action is not completely understood. This study has involved the application of molecular modeling methods and formalisms of computer-aided molecular design (CAMD) in order to explore the interaction between 4-NC and MMP-2/MMP-9, and to design new inhibitors for these targets. Exploratory data analysis, which comprises hierarchical cluster analysis and principal components analysis, was performed to a set of hydroxamates (N=64). previously reported as MMP-2 and MMP-9 inhibitors, in order lo identify the molecular properties that is most critical for the discrimination process regarding the investigated compounds. The thermodynamic, electronic, and steric properties were: quite important to describe the highly active compounds in the data set of MMP-2, whereas the apparent partition coefficient (ClogD) at pH 1.5 was the property more relevant for MMP-9 data set. The presence of bulky substituents on the R3 moiety seems to be crucial for this set of inhibitors due to the molecular interaction with the S1 subsite of both enzymes. However, there is a limit regarding the substituents volume in this region. Receptor independent (RI) 4D-QSAR analysis was applied lo the same data set and it was possible to establish the pharmacophore mapping, besides to explore different alignments in order to generate the hypothesized bioactive conformation through the best QSAR model. The QSAR models have presented good predictability, assisted in proposing new inhibitors, and estimated the activity of 4-NC. Regarding the best QSAR model for MMP-9 (N=64), the 4-NC predicted activity was classified in the range of the moderate active inhibitors. The best QSAR model obtained for MMP-2 (N=38), however was not able to properly predict the activity for compounds with different chemical scaffold from those used to build up the QSAR model. Molecular docking studies have been developed to investigate the 4-NC binding mode into the catalytic site of the two enzymes and the interactions that could be established in those complexes. The results have shown two favorable conformers regarding the MMP inhibition. Molecular dynamics computational simulation were combined to molecular docking studies in order to obtain more detailed and reliable information regarding the intermolecular interactions of each complex. The 4-NC molecule tends to accommodate the side chain in the S1 pocket adjacent to the catalytic site in both enzymes. Experimental zymography assays were also performed to elucidate the possible contribution of the side chain and the catechol core in the 4-NC inhibitory activity against the MMP-2 and MMP-9 enzymes. The catechol core seems to be responsible for its activity, since the 1,2 dimethoxybenzene compound, which has the hydroxyl blocked by a methyl group, was not able to exert any significant inhibition on enzymes. Voltametric assays confirmed the hypothesis that 4-NC chelates zinc ions present in the incubation buffer.

A enzima diidroorotato desidrogenase (DHODH) catalisa a conversão de diidroorotato em orotato, a quarta e única reação redox da via metabólica da síntese de novo de nucleotídeos de pirimidina. DHODH tem sido explorada como alvo validado para terapias contra doenças proliferativas e parasitárias e, em particular, tem sido considerada um alvo atraente para o planejamento de fármacos com ação contra tripanossomatídeos, como parasitos dos gêneros Trypanosoma e Leishmania, que conjuntamente são responsáveis por doenças e mortes que acometem milhões de pessoas em todo o mundo. Neste trabalho, através da combinação de técnicas de DNA recombinante, termofluor, cristalografia de raios-X e ensaios de inibição in vitro e in silico, foi possível identificar sítios alvos na estrutura da DHODH para o desenvolvimento de ligantes, identificar inibidores potentes e seletivos contra as DHODHs de Leishmania major e Trypanosoma cruzi e, caracterizar seus mecanismos de inibição. Finalmente, o efeito leishmanicida observado em nossos ensaios anti-promastigota e os baixos níveis de citotoxicidade observados em células de mamíferos sugerem que alguns dos compostos identificados durante o desenvolvimento deste projeto como potentes inibidores da enzima DHODH poderão ser utilizados como protótipos para o desenvolvimento de fármacos com ação leishmanicida e tripanocida. Combinados, nossos resultados forneceram uma nova e importante contribuição para a compreensão do mecanismo de ação das enzimas DHODH da classe 1A e para o desenho de fármacos baseado nas estruturas das enzimas diidroorotato desidrogenase de Leishmania major e Trypanosoma cruzi. Além disso, a alta identidade sequencial e estrutural observada entre as enzimas de tripanossomatídeos sugerem que uma única estratégia para o desenho de inibidores baseado em estrutura poderá ser usada para explorar a enzima DHODH como alvo terapêutico para várias doenças negligenciadas tropicais como Leishmaniose, Doença do sono e Doença de Chagas / Dihydroorotate dehydrogenase (DHODH) catalyses the conversion of dihydroorote to orotate, the fourth step and only redox reaction in the de novo pyrimidine biosynthetic pathway. DHODH has been exploited as a validated target for therapy against proliferative and parasitic diseases, and in particular, has been considered to be an attractive target for drug development against trypanosomatids, such as parasites from the genera Leishmania and Trypanosoma that collectively cause disease and death in millions of humans. In this work, by combining recombinant DNA technology, thermofluor, X-ray crystallography and in vitro and in silico inhibition assays, we have been able to identify target sites for ligand design, identify potent and selective inhibitors against trypanosomatid DHODHs and fully characterize their mechanism of inibition. Finally, the anti-leishmanial effect observed in our anti-promastigote assays and the low citotoxicity levels observed against mammaliam cells strongly suggest that some of the compounds identified during the development of this project as potent DHODH inhibitors can be used as prototytes for the development of anti-leishmania and anti-trypanosoma drugs. Altogether, our findings provide a new and important contribution to the understanding of the mechanism of action of class 1A DHODHs and for the structure-assisted design of inhibitors against trypanosomatid DHODHs. Furthermore, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to exploit DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping Sickness and Chagas\' Disease.

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.

Métodos computacionais são usados para gerar estruturas de complexo proteína-ligante e estimar suas afinidades. Esse trabalho investigou como as diferentes representações da flexibilidade proteica afetam as poses obtidas por ancoragem molecular e as afinidades atribuídas a essas poses. Os mutantes L99A e L99A/M102Q da lisozima T4 foram escolhidos como sistemas modelo. Um descritor para predição de afinidades baseado na aproximação de energia de interação linear (LIE) foi parametrizado especificamente para ligantes da lisozima e foi usado para estimar as afinidades. A proteína foi representada como um grupo de estruturas cristalográficas ou de estruturas de trajetória de dinâmica molecular. O campo de força OPLS-AA para modelar a proteína e os ligantes e a aproximação de Born generalizada para modelar o solvente foram empregados. O descritor de afinidades parametrizado resultou em desvios médios entre afinidades experimentais e calculadas de 1,8 kcal/mol para um conjunto de testes. O descritor teve desempenho satisfatório na separação entre poses cristalográficas e poses falso-positivo e na identificação de poses falso-positivo. Experimentos de agrupamento de complexos realizados com o objetivo de reduzir o custo computacional para estimar afinidades apresentaram resultados insatisfatórios. As melhores aproximações da teoria do ligante implícito propostas aqui para estimar afinidades consideram conjuntos de estruturas de receptor com o mesmo peso. Configurações de ligante também apresentam o mesmo peso ou são dominadas por uma única configuração. A representação da flexibilidade requer um tratamento estatístico adequado para estimativa de afinidades. Aqui, a associação entre LIE e a teoria do ligante implícito mostrou-se frutífera. / Computational methods are used to generate protein-ligand complex structures and estimate their binding affinities. This work investigated how different representations of protein flexibility affect poses obtained by molecular docking and the affinities attributed to these poses. T4 lysozyme mutants L99A and L99A/M102Q were chosen as model systems. A descriptor for prediction of affinities based on linear interaction energy (LIE) approximation was parametrized specifically to lysozyme ligands and was used to estimate affinities. The protein was represented as a group of crystal structures or as structures from a molecular dynamics trajectory. OPLS-AA force field was used to model protein and ligands and the Generalized Born approximation was used to model solvent. The parametrized affinity descriptor resulted in average deviations between experimental and calculated affinities of 1.8 kcal/mol for a test set. Descriptor performance was satisfactory in the separation between crystal poses and false-positive ones and in the identification of false-positive poses. Clustering of complexes was tried out to reduce computational cost to estimate affinities, but results were poor. The best approximations to the implicit ligand theory proposed here in order to estimate affinities consider groups of receptor structures with the same weight. Ligand configurations also have the same weight or are dominated by only one configuration. The representation of protein flexibility requires an adequate statistical treatment when used to estimate affinities. Here, the linking between LIE and the implicit ligand theory proved itself useful.

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.