A doença de Chagas é uma infecção causada pelo protozoário intracelular Trypanosoma cruzi. Atualmente 7 a 8 milhões de pessoas encontram-se infectadas, e há 25 milhões de pessoas em áreas de risco de contaminação. A cada ano ocorrem 56.000 novos casos e, aproximadamente, 12.000 mortes por complicações oriundas da doença. É endêmica em 21 países da América do Sul, e pode ser encontrada, também, na América do Norte e Europa devido a processos migratórios. Somente dois fármacos estão disponíveis para o tratamento da doença de Chagas, o nifurtimox e o benznidazol, que são ativos somente na fase aguda e causam sérios efeitos adversos. Diante deste panorama, é eminente a necessidade de novos antichagásicos. A enzima cruzaína é a principal cisteíno-protease presente no T. cruzi, é importante para a sobrevivência, diferenciação e entrada do parasita no hospedeiro, se apresentando como um excelente alvo biológico na busca de novos quimioterápicos. Derivados de semicarbazona, tais como o nitrofural e o hidroximetilnitrofural demonstraram atividade inibitória da cruzaína, sendo considerados protótipos na busca de antichagásicos. Utilizando estratégias modernas de planejamento de fármacos por meio da integração entre técnicas computacionais, modelagem molecular e docking, e experimentais, síntese e ensaios biológicos, realizou-se neste trabalho o planejamento, síntese e avaliação biológica de bioisósteros do nitrofural como candidatos à antichagásicos. Aplicou-se estudos de modelagem molecular e docking para 10 compostos derivados de aminoguanidina, semi e tiossemicarbazona; observamos nesses estudos que os compostos contendo tiossemicarbazona apresentaram resultados mais favoráveis ao mecanismo de ação proposto, o qual sugere-se um ataque nucleofílico do resíduo de Cys25 presente no sítio catalítico da enzima cruzaína à tiocarbonila presente nesses compostos. Obtiveram-se através da síntese, 6 compostos caracterizados por RMN 1H e 13C. Tais compostos foram submetidos a ensaios de inibição da cruzaína, sendo que os derivados 6, 9 e 10, apresentaram um perfil de inibição favorável em dose de 100 µM, com valores entre 70 e 75% de inibição. Em ensaio de inibição de crescimento celular em formas epimastigotas do T. cruzi o composto 9 apresentou um IC50 de 19,8 µM, sendo o melhor protótipo para desenvolvimento de um novo agente antichagásico. De uma maneira geral os resultados obtidos nos ensaios biológicos corroboram com os dados apresentados na modelagem molecular, uma vez que os compostos contendo a cadeia lateral tiossemicarbazona mostraram melhores resultados em ambos os testes, demonstrando que a integração entre técnicas computacionais e experimentais se apresenta como uma excelente estratégia na busca de novos agentes antichagásicos. / Chagas disease is an infection caused by the intracellular protozoan Trypanosoma cruzi. Currently 7 million to 8 million people are infected, and there are 25 million people in areas at risk of contamination, with 56,000 new cases each year and roughly 12,000 deaths are related to Chagas complications. It is endemic in 21 countries in South America, and can also be found in North America and Europe due to migration processes. Only two drugs are available for treatment of Chagas disease, nifurtimox and benznidazole, which are active only in the acute phase and cause serious adverse effects. Against this background, it is imminent need for new antichagasic. The enzyme cruzain is the major cysteine protease present in the T. cruzi, is important for the survival, differentiation and entry of the parasite in the host, presenting itself as an excellent biological target in the search for new chemotherapeutic agents. Semicarbazone derivatives, such as nitrofurazone and hydroxymethylnitrofurazone showed inhibitory activity cruzain being considered prototypes in search antichagasic. Using modern drug design strategies through the integration of computational techniques, molecular modeling and docking, and experimental synthesis and biological assays. In this work were performed design, synthesis and biological evaluation of the bioisosters nitrofurazone as candidates for antichagasic. Were applied molecular modeling and docking studies for ten derivatives compounds of aminoguanidine, semi and thiosemicarbazone. In these studies thiosemicarbazone derivatives compounds showed more favorable for the mechanism of action proposed, that suggest a nucleophilic attack of the Cys25 residue present in the catalytic site of the enzyme cruzain in the thiocarbonyl group. Six compounds were synthesized and characterized by 1H and 13C NMR. These compounds were tested for inhibition of cruzain, and derivatives 6, 9 and 10 showed favorable enzyme inhibition at single dose of 100 µM, with values between 70 and 75%. In the inhibition assay of cell growth in epimastigotes forms of T. cruzi, the compound 9 showed an IC50 of 19.8 µM, the best prototype for the development of a new antichagasic agent. In general the results obtained by biological assay corroborate the data presented in molecular modeling, since compounds containing side chain thiosemicarbazone showed better results in both tests, showing that the integration of experimental and computational techniques is presented as a excellent strategy in the search for new agents antichagasic

O câncer cervical afeta milhões de mulheres em todo o mundo a cada ano. A maioria dos casos de câncer cervical é causada pelo vírus do papiloma humano (HPV) que é sexualmente transmissível. Cerca de 40 tipos de HPV infectam o colo do útero e estes são designados como sendo de alto ou de baixo risco com base no seu potencial para provocar lesões de alto grau e câncer. A oncoproteína E7 do HPV está diretamente envolvida no aparecimento de câncer de colo do útero. Esta se associada com a proteína pRb e outros alvos celulares que promovem a imortalização celular e carcinogênese. Apesar de muito progresso nos estudos sobre os HPVs de alto risco, ainda não existe uma terapêutica adequada para o tratamento das lesões e câncer causados por este vírus. Este trabalho teve como objetivo entender as diferenças estruturais entre E7 de alto e baixo risco e sugerir, através de análises de bioinformática, possíveis sítios de ligação e inibidores para a E7. Esta é a primeira descrição da modelagem e análise de dinâmica molecular de quatro estruturas tridimensionais completas da E7 dos tipos de alto risco (HPV tipos 16 e 18), de baixo risco (HPV tipo 11) e não relacionadas ao câncer cervical (HPV tipo 1A). Os modelos foram construídos por uma abordagem híbrida usando modelagem por homologia e ab initio. Os modelos foram usados em simulações de dinâmica molecular por 50 ns, sob condições normais de temperatura e pressão. A desordem intrínseca da sequência da proteína E7 foi avaliada com o uso de ferramentas in silico. Os domínios N-terminal de todas as E7 estudadas, mesmo as de alto risco, exibiram estruturas secundárias depois da modelagem. Nas análises da trajetória da dinâmica molecular, as E7s dos HPVs dos tipos 16 e 18 apresentaram maior instabilidade nos seus domínios N-terminais em relação aos do HPV dos tipos 11 e 01. No entanto, esta variação não afetou a conformação das estruturas secundárias durante a simulação. A análise com ANCHOR indicou que as regiões CR1 e CR2 regiões dos tipos de HPV 16 e 18 contêm possíveis alvos para a descoberta da droga. Já a região CR3 do domínio C-terminal indicou estabilidade nas análises in silico e, por isso, foi usada como alvo de busca de modelos farmacofóricos e docking macromolecular. A proteína usada como modelo foi a E7 do HPV tipo 45 resultante de análises de ressonância magnética nuclear (RMN) e depositada no banco de dados de proteína (ID: 2F8B). Foram selecionados por análises sequenciais de busca farmacofórica, docking e re-docking, 19 compostos (extraídos de amplas bibliotecas de pequenos ligantes) com potencial para candidatos a inibidores da E7. Eles foram avaliados quanto a sua função de pontuação, mapas de interação receptor-ligante e toxicidade e os melhores foram indicados para estudos futuros. / Cervical cancer affects millions of women around the world each year. Most cases of cervical cancer are caused by human papilloma virus (HPV) which is sexually transmitted. About 40 types of HPV infect the cervix and these are designated as being at high or low risk based on their potential to cause high-grade lesions and cancer. The E7 oncoprotein from HPV is directly involved in the onset of cervical cancer. It associates with the pRb protein and other cellular targets that promote cell immortalization and carcinogenesis. Although the progress in studies with high-risk HPVs there is still no adequate therapy for the treatment of lesions and cancers caused by this virus. This study aimed to understand the structural differences between E7 of high and low risk and suggest, with the aid of bioinformatics analyzes, possible binding sites and inhibitors for the E7. This is the first description of the modeling and molecular dynamics analysis of four complete three-dimensional structures of E7 from high-risk types (HPV types 16 and 18), low risk (HPV type 11) and that not related to cervical cancer (HPV 01). The models were constructed by a hybrid approach using homology modeling and ab initio. The models were used in molecular dynamics simulations for 50 ns, under normal temperature and pressure. The intrinsic disorder of the E7 protein sequence was assessed using in silico tools. The N-terminal domains of all E7s, even the high-risks, showed secondary structures after modeling. In the trajectory analyzes of molecular dynamics, the E7s of HPV types 16 and 18 showed high instability in their N-terminal domains than those of HPV types 11 and 01, however, this variation did not affect the conformation of secondary structures during the simulation. The analysis with ANCHOR indicated that regions CR1 and CR2 regions of types of HPV 16 and 18 contain possible targets for drug discovery. The CR3 region of the C-terminal domain indicated stability by in silico analyzes and was therefore used as target to search for pharmacophoric models and \"docking\". The protein used as a model was the E7, from HPV type 45, constructed by analysis of nuclear magnetic resonance (NMR) and deposited in the protein data bank (ID: 2F8B). It was selected 19 compounds as potential candidates for E7 inhibitors (extracted from large libraries of small ligands) using sequential pharmacophore search, docking and re-docking analyzes. They were evaluated for their scoring function, maps of receptor-ligand interactions and toxicity and the best suited were indicated for future studies.

Orientador : Kleber Gomes Franchini / Tese (Doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-22T00:30:15Z (GMT). No. of bitstreams: 1 Antunes_JoaoEustaquio_D.pdf: 5472161 bytes, checksum: d84f6389e6baf3e2959126f6345e6a14 (MD5) Previous issue date: 2013 / Resumo: A compreensão de como a quinase de adesão focal (FAK) contribui para os processos de hipertrofia, insuficiência cardíaca e câncer são de grande interesse científico. Um dos nossos objetivos específicos é desenvolver inibidores desta tirosina quinase com vistas à sua aplicação terapêutica no tratamento de insuficiência cardíaca e câncer. Portanto, foi realizado planejamento racional e a síntese de inibidores farmacológicos para a FAK. Estudos computacionais de docking e farmacocinéticos permitiram selecionar 28 estruturas de quinazolina mais promissoras em relação à capacidade de inibir a FAK. Desta forma, economiza-se tempo e dinheiro para obter-se a síntese apenas de 6 das estruturas pré-selecionadas. Uma quinazolina denominada 4-BZLO foi sintetizada após o planejamento racional. Tal quinazolina foi capaz de inibir em 50% da atividade da FAK in vitro com aproximadamente 1nM. Os testes de pureza da síntese, absorção por via oral, melhor resultado em triagem em células que superexpressam a FAK e o melhor resultado para triagem em duas linhagens de célula leucêmicas e tumor sólido permitiram direcionar o 4- BZLO para ser o composto líder deste estudo. O composto 4-BZLO apresentou resultado promissor para experimentos com camundongos submetidos à coarctação da aorta, que induz hipertrofia cardíaca. Para avaliar se o tratamento preventivo seria eficiente para hipertrofia cardíaca, foi realizado experimento em animais tratados com 30mg/Kg/dia durante 30 dias. Após este período, foi realizada cirurgia de coarctação da aorta para induzir a hipertrofia cardíaca nos animais e os mesmos foram tratados por mais 30 dias. Parâmetros tais como: peso médio do ventrículo esquerdo sobre peso corpóreo de cada animal (LVW/BW), medida da espessura da parede do ventrículo esquerdo (LVWT) e parâmetros histológicos (diâmetro dos miócitos cardíacos) demonstrou que nos animais tratados houve regressão da hipertrofia comparada ao controle (animais sem tratamento). Outro estudo realizado no qual os animais foram tratados de maneira curativa, ou seja, o tratamento foi realizado somente após a coarctação da aorta demonstrou uma melhora no quadro de hipertrofia e função cardíaca. O modelo de fibrose cardíaca foi usado para avaliar se tratamento com 4-BZLO é capaz de reduzir a fibrose cardíaca nos animais. Os resultados obtidos demonstraram que os animais tratados com 4-BZLO por 30 dias apresentaram redução da acumulação de colágeno, que é um indicador de fibrose, em relação ao controle. Nosso laboratório desenvolveu camundongos transgênicos específicos para a FAK que desenvolve moderada hipertrofia cardíaca. Assim, tal modelo permitiu testar o tratamento com o 4-BZLO para hipertrofia cardíaca induzida pela FAK. Os animais transgênicos específicos para a FAK foram tratados com o composto líder e houve melhora dos parâmetros cardíacos. Os resultados obtidos permitiram concluir que a quinazolina denominada 4-BZLO é um bom candidato a fármaco como inibidor da FAK / Abstract: Understanding how the focal adhesion kinase (FAK) contributes to the processes of hypertrophy, heart failure and cancer are of great scientific interest. One of our goals is to develop specific inhibitors of this tyrosine kinase with potential therapeutic application in the treatment of heart failure and cancer. In this view, we used rational design to select a group of possible FAK inhibitors. Computational studies such as docking and pharmacokinetic studies allowed us to select 28 structures most likely to inhibit FAK. In this way, we saved up time and money in devising the synthesis of 6 pre-selected structures. Accordingly, quinazoline 4-BZLO was prepared and was able to inhibit the in vitro activity of FAK by 50% at a concentration of approximately 1nM. Several factors contributed to 4-BZLO being chosen as the lead compound in this study: 1) the degree of purity achieved during synthesis; 2) good oral bioavailability; 3) the best inhibition values in cells over expressing FAK in screening, 4) the best result against two leukemic cell lines and one solid tumor target. 4-BZLO showed promising results in experiments with mice subjected to aortic coarctation, which develops cardiac hypertrophy. To assess whether 4- BZLO would be effective for the preventive treatment of cardiac hypertrophy, an experiment was conducted in animals treated with 30 mg/kg/day for 30 days. After this period, an aortic coarctation was performed surgically in order to induce cardiac hypertrophy in animals, and these were further treated for 30 days. Parameters such as left ventricular weight per body weight ratio (LVW/BW), measurement of the left ventricle wall thickness (LVWT), and histological parameters, such as the diameter of cardiac myocytes in treated animals showed that there was a regression of hypertrophy, compared to untreated animals (control). A similar study, where treatment with 4-BZLO was performed only after aortic coarctation showed an improvement regarding hypertrophy and cardiac function. A cardiac fibrosis model was used and the results obtained demonstrated that animals treated with 4-BZLO for 30 days showed a reduction of collagen accumulation, which is an indicator of fibrosis, equal to the control group. Our laboratory has developed transgenic mice specific for FAK, which develop moderate cardiac hypertrophy. Consequently, this model allows us to test 4-BZLO for the treatment of FAK induced cardiac hypertrophy. The transgenic animals were treated with 4-BZLO, leading to an improvement of the cardiac parameters. These results showed that synthetic quinazoline 4-BZLO is a good drug candidate for the inhibition of the FAK enzyme / Doutorado / Farmacologia / Doutor em Farmacologia

Ce travail de thèse consiste au développement méthodologique et logiciel d'une méthode de docking moléculaire dite inverse. Cette méthode propose à travers le programme AMIDE — Automatic Inverse Docking Engine — de distribuer un grand nombres de simulations d'amarrage moléculaire sur des architectures HPC (clusters de calcul) avec les applications AutoDock 4.2 et AutoDock Vina. Le principe de cette méthode consiste à tester de petites molécules sur un ensemble de protéines cibles potentielles. Les paramètres optimaux ont été définis à partir d'une étude pilote et le protocole a été validé sur des ligands et peptides liants les protéines MMPs et EBP de la matrice extracellulaire. Cette méthode montre qu'elle permet d‘améliorer la recherche conformationnelle lors du calcul de docking sur des structures expérimentales par rapport à des protocoles existants (blind docking). Il est montré que le programme AMIDE permet de discriminer des sites de fixation privilégiés lors d'expériences de criblage inverse de protéines de manière plus performante que par blind docking. Ces résultats sont obtenus par la mise en place de méthodes de partitionnement de l'espace de recherche qui permettent également à travers un système de distribution hybride de déployer un ensemble de tâches indépendantes pour un traitement autorisant le passage d'échelle. / This work is a methodological and software development of so-called inverse molecular docking method. This method offers through an in house program AMIDE — Automatic Reverse Docking Engine — to distribute large numbers of molecular docking simulations on HPC architectures (com- puting clusters) with AutoDock 4.2 and AutoDock Vina applications. The principle of this method is to test small molecules on a set of potential target proteins. The program optimum parameters were defined from a pilot study and the protocol was validated on ligands and peptides binding MMPs and EBP extracellular matrix proteins. This method improves the conformational search in docking computation on experimental structures compared to existing protocols (blind docking). It is shown that the AMIDE program is more efficient to discriminate preferred binding sites in inverse proteins screening experiments than blind docking. These results are obtained by the implemen- tation of methods for partitioning the search space that also allow through a hybrid distribution system to deploy a set of independent embarassingly parallel tasks perfectly scalable.

Orientadores: João Ernesto de Carvalho, Mary Ann Foglio / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T11:34:15Z (GMT). No. of bitstreams: 1 Rizzo_LarissaYokota_M.pdf: 2545881 bytes, checksum: 63ab51d7b009706ec8505aa2b3ded262 (MD5) Previous issue date: 2011 / Resumo: A pesquisa de drogas anticâncer através da triagem de extratos e princípios ativos obtidos de fontes naturais possibilitou a descoberta e o desenvolvimento de diversos quimioterápicos hoje utilizados no tratamento do câncer. Este projeto teve como objetivo avaliar a atividade anticâncer de Psidium guajava L. (nome popular: goiabeira), espécie eleita através de levantamento etnofarmacológico para atividade antiparasitária. Após a colheita, o material vegetal foi submetido a um processo de extração a quente por Soxhlet, com diclorometano e etanol 95%, originando o extrato bruto diclorometânico (EBD) e etanólico (EBE), respectivamente. O extrato bruto ativo, EBD, foi submetido a diversos fractionamentos biomonitorados, até a obtenção de uma fração enriquecida nos meroterpenos guajadial, psidial A e seus isômeros, princípios ativos da espécie vegetal. Todas as amostras (extratos brutos e frações enriquecidas) tiveram sua potencial atividade anticâncer avaliadas in vitro frente a um painel de dez linhagens tumorais humanas, cedidas pelo National Cancer Institute (NCI, Estados Unidos), a saber: K562 (leucemia), MCF-7 (mama), NCI/ADR-RES (ovário resistente a múltiplas drogas), NCI-H460 (pulmão), UACC62 (melanoma), PC-3 (próstata), HT-29 (cólon), OVCAR-03 (ovário), U251 (glioma) and 786-0 (rim). A fração ativa enriquecida nos meroterpenos foi avaliada in vivo no tumor sólido de Ehrlich, em camundongos Balb/C, reduzindo significativamente o crescimento tumoral. Além da atividade antitumoral observada in vivo, a análise macroscópica do útero indicou aumento em tamanho e peso em relação aos grupos controle negativo (salina) e positivo (doxorrubicina). As moléculas de guajadial e psidial A apresentam propriedades físico-químicas semelhantes ao estradiol e ao tamoxifeno e estudos de docking molecular sugerem que ambos os compostos se liguem no sítio de ligação no receptor de estrógeno (ER), analogamente ao tamoxifeno. A capacidade de reduzir o crescimento tumoral e, ao mesmo tempo, estimular o útero indicam que guajadial e psidial A possivelmente agem como fitoestrógenos, possuindo um mecanismo de ação semelhante ao tamoxifeno, atuando como SERMs (Selective Estrogen Receptor Modulators) e agindo como agonistas e antagonistas de forma tecido específica / Abstract: Anticancer drug research based on screening of natural sources enabled the discovery of several drugs that are used in cancer treatment. This project aimed to evaluate the in vitro and in vivo anticancer activity of Psidium guajava L. (popular name: guava), elected for its ethnopharmacological use for antiparasitic activity. After harvesting, the vegetal material was extracted in Soxhlet with dicloromethane and then ethanol 95%, leading to the dicloromethane crude extract (DCE) and the ethanolic crude extract (ECE), respectively.The active extract, DCE, was submitted to several biomonitored fractionating processes and an active mixture of meroterpenes identified guajadial, psidial A and its isomers as active principles. All samples (crude extract and enriched fractions) were evaluated in vitro for cytotoxic activity against ten human cancer lines (donated by National Cancer Institute, USA): K562 (leukemia), MCF-7 (breast), NCI/ADR-RES (resistant ovarian cancer), NCI-H460 (lung), UACC62 (melanoma), PC-3 (prostate), HT-29 (colon), OVCAR-03 (ovary), U251 (glioma) and 786-0 (kidney). Meroterpenes enriched fraction was evaluated in vivo in the Solid Ehrlich Tumor, in Balb/C mice, and significantly reduced tumor growth. Besides antitumoral activity, macroscopic analysis of uterus showed increased size and weight in comparison to both negative (vehicle) and positive (doxorubicin) control groups. The molecules of guajadial and psidial A display similar physicochemical properties to estradiol and tamoxifen and in silico molecular docking studies suggest that both molecules bind to the estrogen-binding site of ERs analogously to tamoxifen. The ability to reduce breast cancer tumor growth and stimulate the uterus suggests that guajadial and psidial A may act as phytoestrogens, giving insights for a mechanism of action similar to tamoxifen, acting as SERMs (Selective Estrogen Receptor Modulators), having both agonist and antagonist tissue-specific activities / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Ana Carolina Gomes Jardim / Resumo: Nas últimas décadas, diversos vírus que tinham sua ocorrência limitada a pequenas regiões se espalharam pelo globo, causando epidemias e preocupação entre as autoridades de saúde. Apesar dos inúmeros avanços no tratamento das infecções virais, vários destes vírus ainda não possuem tratamento especifico e eficaz. Adicionalmente, a alta taxa de resistência e o surgimento de novas mutações, torna a busca por novos antivirais desafiadora e de extrema importância. Desta forma, o presente trabalho teve como objetivo investigar a atividade antiviral de compostos de origem natural ou sintética contra os vírus da Chikungunya (CHIKV) e Enterovirus A71 (EV-A71). Contra o CHIKV, 48.750 compostos sintéticos foram inicialmente avaliados in silico por docking molecular, dos quais 12 compostos demonstraram apresentar interação com a região de ligação a ADP-ribose do dominío macro da proteína viral não estrutural 3 (nsP3), e foram selecionados para ensaios in vitro. Ensaios de viabilidade celular foram realizados para determinar a máxima concentração não tóxica de cada composto, que foi utilizada nos ensaios anti-CHIKV em células de hepatocarcimona humano Huh-7, transfectadas com os replicons subgenômicos do CHIKV. Os resultados demonstraram que os compostos C5 e C13 na concentração de 20 µM inibiram 53 e 76% da replicação do CHIKV em células Huh-7, respectivamente. Contra o EV-A71, 6 proteínas isoladas da peçonha de serpentes foram testadas em concentrações não tóxicas em células Vero infect... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the last decades, several viruses that had their occurrence limited to small regions spread through the globe, causing epidemics and concern among health authorities. Despite the numerous advances in the treatment of viral infections, several of these viruses have no specific and effective treatment yet. In addition, the high rate of resistance and the emergence of new mutations, makes the search for new antivirals challenging and extremely important. The present work aimed to investigate the antiviral activity of compounds from natural or synthetic origin against Chikungunya virus (CHIKV) and Enterovirus A71 (EV-A71). Against CHIKV, 48,750 synthetic compounds were initially evaluated in silico by molecular docking, of which 12 compounds demonstrated to be interacting with the ADP-ribose binding region of viral non- structural protein 3 (nsP3) macro domain and were selected for in vitro assays. Cell viability assays were performed to determine the maximum non-toxic concentration of each compound and used in anti-CHIKV assays in human hepatocarcinoma cells (Huh-7) transiently transfected with the CHIKV subgenomics replicons. The results demonstrated that the C5 and C13 compounds at 20 µM inhibited 53 and 76% of CHIKV replication in Huh-7 cells, respectively. Against EV-A71, 6 proteins isolated from snake venom were tested at non-toxic concentrations in infected Vero cells, and the virucidal, protective and anti-EV-A71 replication activity was evaluated. From the tested toxi... (Complete abstract click electronic access below) / Doutor

Molecular docking has been extensively applied in virtual screening of small molecule libraries for lead identification and optimization. A necessary prerequisite for successful differentiation between active and non-active ligands is the accurate prediction of their binding affinities in the complex by use of docking scoring functions. However, many studies have shown rather poor correlations between docking scores and experimental binding affinities. Our work aimed to improve this correlation by implementing a multipose binding concept in the docking scoring scheme. Multipose binding, i.e., the property of certain protein-ligand complexes to exhibit different ligand binding modes, has been shown to occur in nature for a variety of molecules. We conducted a high-throughput docking study and implemented multipose binding in the scoring procedure by considering multiple docking solutions in binding affinity prediction. In general, improvement of the agreement between docking scores and experimental data was observed, and this was most pronounced in complexes with large and flexible ligands and high binding affinities. Further developments of the selection criteria for docking solutions for each individual complex are still necessary for a general utilization of the multipose binding concept for accurate binding affinity prediction by molecular docking.

Molecular interaction among proteins drives the cellular processes through the formation of complexes that perform the requisite biochemical function. While some of the complexes are obligate (i.e., they fold together while complexation) others are non-obligate, and are formed through macromolecular recognition. Macromolecular recognition in proteins is highly specific, yet it can be both permanent and non permanent in nature. Hallmarks of permanent recognition complexes include large surface of interaction, stabilization by hydrophobic interaction and other noncovalent forces. Several amino acids which contribute critically to the free energy of binding at these interfaces are called as “hot spot” residues. The non permanent recognition complexes, on the other hand, usually show small interface of interaction, with limited stabilization from non covalent forces. For both the permanent and non permanent complexes, the specificity of molecular interaction is governed by the geometric compatibility of the interaction surface, and the noncovalent forces that anchor them. A great deal of studies has already been performed in understanding the basis of protein macromolecular recognition.1; 2 Based on these studies efforts have been made to develop protein-protein docking algorithms that can predict the geometric orientation of the interacting molecules from their individual unbound states. Despite advances in docking methodologies, several significant difficulties remain.1 Therefore, in this thesis, we start with literature review to understand the individual merits and demerits of the existing approaches (Chapter 1),3 and then, we attempt to address some of the problems by developing methods to infer protein quaternary structure from the crystalline state, and improve structural and chemical understanding of protein-protein interactions through biological complex prediction. The understanding of the interaction geometry is the first step in a protein-protein interaction study. Yet, no consistent method exists to assess the geometric compatibility of the interacting interface because of its highly rugged nature. This suggested that new sensitive measures and methods are needed to tackle the problem. We, therefore, developed two new and conceptually different measures using the Delaunay tessellation and interface slice selection to compute the surface complementarity and atom packing at the protein-protein interface (Chapter 2).4 We called these Normalized Surface Complementarity (NSc) and Normalized Interface Packing (NIP). We rigorously benchmarked the measures on the non redundant protein complexes available in the Protein Data Bank (PDB) and found that they efficiently segregate the biological protein-protein contacts from the non biological ones, especially those derived from X-ray crystallography. Sensitive surface packing/complementarity recognition algorithms are usually computationally expensive and thus limited in application to high-throughput screening. Therefore, special emphasis was given to make our measure compute-efficient as well. Our final evaluation showed that NSc, and NIP have very strong correlation among themselves, and with the interface area normalized values available from the Surface Complementarity program (CCP4 Suite: <http://smb.slac.stanford.edu/facilities/software/ccp4/html/sc.html>); but at a fraction of the computing cost. After building the geometry based surface complementarity and packing assessment methods to assess the rugged protein surface, we advanced our goal to determine the stabilities of the geometrically compatible interfaces formed. For doing so, we needed to survey the quaternary structure of proteins with various affinities. The emphasis on affinity arose due to its strong relationship with the permanent and non permanent life-time of the complex. We, therefore, set up data mining studies on two databases named PQS (Protein Quaternary structure database: http://pqs.ebi.ac.uk) and PISA (Protein Interfaces, Surfaces and Assemblies: www.ebi.ac.uk/pdbe/prot_int/pistart.html) that offered downloads on quaternary structure data on protein complexes derived from X-ray crystallographic methods. To our surprise, we found that above mentioned databases provided the valid quaternary structure mostly for moderate to strong affinity complexes. The limitation could be ascertained by browsing annotations from another curated database of protein quaternary structure (PiQSi:5 supfam.mrc-lmb.cam.ac.uk/elevy/piqsi/piqsi_home.cgi) and literature surveys. This necessitated that we at first develop a more robust method to infer quaternary structures of all affinity available from the PDB. We, therefore, developed a new scheme focused on covering all affinity category complexes, especially the weak/very weak ones, and heteromeric quaternary structures (Chapter 3).6 Our scheme combined the naïve Bayes classifier and point-group symmetry under a Boolean framework to detect all categories of protein quaternary structures in crystal lattice. We tested it on a standard benchmark consisting of 112 recognition heteromeric complexes, and obtained a correct recall in 95% cases, which are significantly better than 53% achieved by the PISA,7 a state-of-art quaternary structure detection method hosted at the European Bioinformatics Institute, Hinxton, UK. A few cases that failed correct detection through our scheme, offered interesting insights into the intriguing nature of protein contacts in the lattice. The findings have implications for accurate inference of quaternary states of proteins, especially weak affinity complexes, where biological protein contacts tend to be sacrificed for the energetically optimal ones that favor the formation/stabilization of the crystal lattice. We expect our method to be used widely by all researchers interested in protein quaternary structure and interaction. Having developed a method that allows us to sample all categories of quaternary structures in PDB, we set our goal in addressing the next problem that of accurately determining stabilities of the geometrically compatible protein surfaces involved in interaction. Reformulating the question in terms of protein-protein docking, we sought to ask how we could reliably infer the stabilities of any arbitrary interface that is formed when two protein molecules are brought sterically closer. In a real protein docking exercise this question is asked innumerable times during energy-based screening of thousands of decoys geometrically sampled (through rotation+translation) from the unbound subunits. The current docking methods face problems in two counts: (i), the number of interfaces from decoys to evaluate energies is rather large (64320 for a 9º rotation and translation for a dimeric complex), and (ii) the energy based screening is not quite efficient such that the decoys with native-like quaternary structure are rarely selected at high ranks. We addressed both the problems with interesting results. Intricate decoy filtering approaches have been developed, which are either applied during the search stage or the sampling stage, or both. For filtering, usually statistical information, such as 3D conservation information of the interfacial residues, or similar facts is used; more expensive approaches screen for orientation, shape complementarity and electrostatics. We developed an interface area based decoy filter for the sampling stage, exploiting an assumption that native-like decoys must have the largest, or close to the largest, interface (Chapter 4).8 Implementation of this assumption and standard benchmarking showed that in 91% of the cases, we could recover native-like decoys of bound and unbound binary docking-targets of both strong and weak affinity. This allowed us to propose that “native-like decoys must have the largest, or close to the largest, interface” can be used as a rule to exclude non native decoys efficiently during docking sampling. This rule can dramatically clip the needle-in-a-haystack problem faced in a docking study by reducing >95% of the decoy set available from sampling search. We incorporated the rule as a central part of our protein docking strategy. While addressing the question of energy based screening to rank the native-like decoys at high rank during docking, we came across a large volume of work already published. The mainstay of most of the energy based screenings that avoid statistical potential, involve some form of the Coulomb’s potential, Lennard Jones potential and solvation energy. Different flavors of the energy functions are used with diverse preferences and weights for individual terms. Interestingly, in all cases the energy functions were of the unnormalized form. Individual energy terms were simply added to arrive at a final score that was to be used for ranking. Proteins being large molecules, offer limited scope of applying semi-empirical or quantum mechanical methods for large scale evaluation of energy. We, therefore, developed a de novo empirical scoring function in the normalized form. As already stated, we found NSc and NIP to be highly discriminatory for segregating biological and non biological interface. We, therefore, incorporated them as parameters for our scoring function. Our data mining study revealed that there is a reasonable correlation of -0.73 between normalized solvation energy and normalized nonbonding energy (Coulombs + van der Waals) at the interface. Using the information, we extended our scoring function by combining the geometric measures and the normalized interaction energies. Tests on 30 unbound binary protein-protein complexes showed that in 16 cases we could identify at least one decoy in top three ranks with ≤10 Å backbone root-mean-square-deviation (RMSD) from true binding geometry. The scoring results were compared with other state-of-art methods, which returned inferior results. The salient feature of our scoring function was exclusion of any experiment guided restraints, evolutionary information, statistical propensities or modified interaction energy equations, commonly used by others. Tests on 118 less difficult bound binary protein-protein complexes with ≤35% sequence redundancy at the interface gave first rank in 77% cases, where the native like decoy was chosen among 1 in 10,000 and had ≤5 Å backbone RMSD from true geometry. The details about the scoring function, results and comparison with the other methods are extensively discussed in Chapter 5.9 The method has been implemented and made available for public use as a web server - PROBE (http://pallab.serc.iisc.ernet.in/probe). The development and use of PROBE has been elaborated in Chapter 7.10 On course of this work, we generated huge amounts of data, which is useful information that could be used by others, especially “protein dockers”. We, therefore, developed dockYard (http://pallab.serc.iisc.ernet.in/dockYard) - a repository for protein-protein docking decoys (Chapter 6).11 dockYard offers four categories of docking decoys derived from: Bound (native dimer co-crystallized), Unbound (individual subunits as well as the target are crystallized), Variants (match the previous two categories in at least one subunit with 100% sequence identity), and Interlogs (match the previous categories in at least one subunit with ≥90% or ≥50% sequence identity). There is facility for full or selective download based on search parameters. The portal also serves as a repository to modelers who may want to share their decoy sets with the community. In conclusion, although we made several contributions in development of algorithms for improved protein-protein docking and quaternary structure inference, a lot of challenges remain (Chapter 8). The principal challenge arises by considering proteins as flexible bodies, whose conformational states may change on quaternary structure formation. In addition, solvent plays a major role in the free energy of binding, but its exact contribution is not straightforward to estimate. Undoubtedly, the cost of computation is one of the limiting factors apart from good energy functions to evaluate the docking decoys. Therefore, the next generation of algorithms must focus on improved docking studies that realistically incorporate flexibility and solvent environment in all their evaluations.

Molecular docking has been extensively applied in virtual screening of small molecule libraries for lead identification and optimization. A necessary prerequisite for successful differentiation between active and non-active ligands is the accurate prediction of their binding affinities in the complex by use of docking scoring functions. However, many studies have shown rather poor correlations between docking scores and experimental binding affinities. Our work aimed to improve this correlation by implementing a multipose binding concept in the docking scoring scheme. Multipose binding, i.e., the property of certain protein-ligand complexes to exhibit different ligand binding modes, has been shown to occur in nature for a variety of molecules. We conducted a high-throughput docking study and implemented multipose binding in the scoring procedure by considering multiple docking solutions in binding affinity prediction. In general, improvement of the agreement between docking scores and experimental data was observed, and this was most pronounced in complexes with large and flexible ligands and high binding affinities. Further developments of the selection criteria for docking solutions for each individual complex are still necessary for a general utilization of the multipose binding concept for accurate binding affinity prediction by molecular docking.

Un des principaux défis du domaine de la biologie structurale est l'obtention d'informations à haute résolution sur les grandes macromolécules biologiques. En raison de leurs tailles et de leurs flexibilités, les techniques traditionnelles de biologie structurales sont souvent impuissantes. Une des techniques prometteuses est la microscopie à force atomique (AFM). Contrairement à la microscopie optique, l'AFM utilise une sonde mécanique de très faible taille (<10 nm) pour obtenir des informations topographiques sur du matériel biologique isolé et déposé sur des surfaces ultras plates. L'objectif du travail de thèse est de développer les outils informatiques pour permettre la modélisation de grandes macromolécules au niveau atomique tout en intégrant des contraintes topologiques obtenues par l'imagerie AFM. À partir d'images AFM de hauteur, à haute résolution, un protocole d'assemblage de domaines protéiques a été mis au point. Il utilise une recherche exhaustive dans l'espace tridimensionnel réel de toutes les orientations possibles des domaines de la macromolécule à modéliser qui respectent les contours imposés par l'image AFM. Un jeu de contraintes de distance entre chacun des domaines permet un premier tri des modèles candidats. Un classement final est attribué à chaque modèle selon un score appelé EFactor, estimateur de la ressemblance entre la surface topographique expérimentale et celle du modèle. Le protocole a été validé sur le système modèle que sont les anticorps. Il a été également utilisé pour reconstruire une particule virale (virus de la mosaïque du tabac) et assembler la structure tétramérique de la protéine membranaire l'aquaporine Z. / A major challenge in the field of structural biology is to obtain high-resolution information on the major biological macromolecules. Because of their size and their flexibility, the traditional techniques of structural biology are often powerless. One of the promising techniques is atomic force microscopy (AFM). Unlike optical microscopy, AFM uses a mechanical probe of very small size (<10 nm) to obtain topographical information on isolated biological material deposited on ultra flat surfaces. The aim of the thesis was to develop tools to enable the modeling of large macromolecules at the atomic level while incorporating topological constraints obtained by AFM imaging. Using high resolution AFM height images, a protocol for assembling protein domains has been developed. It uses an exhaustive search in real three-dimensional space of all possible orientations of the macromolecule's domains respecting the boundaries imposed by the AFM topographical image. A set of distance constraints between each of the domains allows an initial screening of candidate models. A final ranking is assigned to each model according to a score called EFactor, estimator of the similarity between the experimental topography and the model. The protocol was validated on model systems that are antibodies. It was also used to reconstruct a virus particle (tobacco mosaic virus) and assemble the tetrameric structure of the membrane protein aquaporin Z.