Submitted by FABIANA DA SILVA FRANÇA (fabiana21franca@gmail.com) on 2017-11-09T14:11:12Z No. of bitstreams: 2 arquivototal.pdf: 4984003 bytes, checksum: 15bf30b6c475733d92d8611db7b64bc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-09T14:11:12Z (GMT). No. of bitstreams: 2 arquivototal.pdf: 4984003 bytes, checksum: 15bf30b6c475733d92d8611db7b64bc0 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-11-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Ricinus communis, specifically fruit, castor bean, has gained industry, media and government attention due to the derivate properties such as ricin and castor oil. The total fatty acids xtracted from castor bean, 90% comprises ricinoleic acid. Beyond the castor oil, castor bean processing generate co-products: fruit peel and the castor cake. These co-products present significant amounts of fibers and proteins, in addition to a potential use as nematicide. Since world production of castor oil is 1.5 million tons per year and the proportion of co-product production with castor oil production is approximately 1:1, so, it’s essential to find an economically viable destination for these co-products. In addition to the destinations used, a route with high commercial value would be the use as animal feed, however, this does not occur due to the presence of ricin. Ricin its a ribosome inactivating protein present in the castor bean seed, that consists of two subunits, RTA and RTB, with the RTA being the catalytic subunit. In addition this problem, ricin is used as a biological weapon by terrorists and activists. The inhibition of mechanism of ricin action has biotechnological interest, where RTA is the target for inhibitors synthesis. In this context, the method used to search for new inhibitors its Molecular Docking. This method evaluates thousands of ligands in a short time, however, presents low accuracy in the prediction of binding affinity. In this perspective, SMD simulations can be used. This method is based on the correlation of the mean force profile needed to decouple the ligand from the protein with its affinity. The scientific literature has reported promising results with the approach to discern active binders from inactive. In the present study, beyond the validation data from known RTA inhibitors, the binding affinity potential of 6 novel structures to form complexes with RTA using SMD simulations was evaluated. It was necessary to use Molecular Docking and Molecular Dynamics aproaches to obtain and refine new RTA complexes with novel ligands and next submitted to SMD simulation (k = 2 kcal/mol/Ų, v = 0.005 nm/ps). In addition to the mean force profile obtained from multiple independent SMD simulations, the rupture force and the average of pulling work were measured, these being a direct relation with the binding affinity. In summary, the results of the validation of rupture force and average of pulling work with the experimental data showed a correlation and determination coefficient with R = -0.992 and R² = 0.984 (rupture force) and R -0.958 and R² = 0.918 (average of pulling work), respectively. By means of these validations, the evaluation of novel structures using SMD simulations showed that 4 of the 6 proposed structures present in silico, binding affinity potential for RTA. / A Ricinus communis, especificamente o seu fruto, a mamona, tem ganhado atenção da indústria, mídia e governos, devido as propriedades de seus derivados, especialmente a ricina e o óleo. Do total de ácidos graxos extraídos da mamona, 90% compreende ao ácido ricinoleico. Além do óleo, os coprodutos gerados durante a produção tem ganhado bastante atenção, sendo os mais importantes, a casca do fruto e a torta. Tais coprodutos apresentam quantidades significativas de fibras e proteínas, além de um potencial uso como nematicida. Uma vez que a produção mundial de óleo de mamona é de 1,5 milhão de toneladas-ano e a proporção de produção de coprodutos com a produção de óleo é de aproximadamente 1:1, é fundamental encontrar um destino economicamente viável para esses coprodutos. Além dos destinos utilizados, uma rota com alto valor comercial seria o uso como ração animal, entretanto, isto não ocorre devido a presença da ricina. Esta, corresponde a uma proteína inativadora de ribossomos presente na semente da mamona, e é constituída por duas subunidades, a RTA e RTB, sendo a RTA a subunidade catalítica. Além da problemática supracitada, a ricina é utilizada como arma biológica por terroristas e ativistas, logo, a inibição do mecanismo de ação desta proteína é de grande interesse biotecnológico, sendo a RTA o alvo para síntese de inibidores. Neste aspecto, o método utilizado para busca de novos inibidores é o Atracamento Molecular (Molecular Docking). Este método avalia milhares de ligantes num curto intervalo de tempo, entretanto, apresenta baixa acurácia na predição da afinidade de ligação. Nesta perspectiva, a Dinâmica Molecular Induzida (SMD), pode ser utilizada. Este método baseia-se na correlação do perfil de força médio necessário para desacoplar o ligante da proteína com a sua afinidade. A literatura científica tem relatado resultados promissores no uso dessa abordagem para discernir ligantes ativos de inativos. No presente estudo, além de validações com inibidores de RTA conhecidos, foi avaliado o potencial de afinidade de 6 estruturas inéditas a formarem complexos com a RTA utilizando simulações SMD. Para isso, foi necessário recorrer a abordagens de Atracamento Molecular e Dinâmica Molecular para obtenção e refinamento de novos complexos da RTA com os ligantes candidatos, para somente então, serem submetidos a simulação SMD (k = 2 kcal/mol/Ų, v = 0.005 nm/ps). Neste passo, além do perfil de força médio obtido a partir de múltiplas simulações SMD independentes, foi mensurado a força de ruptura e o trabalho médio realizado pela força, este último, apresenta uma relação direta com a afinidade de ligação pela igualdade de Jarzynski. Em suma, resultados de validação da força de ruptura e trabalho com os dados experimentais mostraram coeficiente de correlação e determinação com R = -0.992 e R² = 0.984 (força de ruptura) e R -0.958 e R² = 0.918 (perfil médio do trabalho), respectivamente. Por meio destas validações, a avaliação dos ligantes candidatos utilizando simulações SMD mostrou que 4 das 6 estruturas propostas, apresentam in silico, potencial de afinidade de ligação para com a RTA.

Les anticorps thérapeutiques sont couramment utilisés pour le traitement contre le cancer. Ils sont sélectionnées par leur affinité avec leur antigène mesuré normalement dans un environnent à trois dimensions (3D). Cependant, de fois les interactions anticorps-antigène ont lieu à l’interface entre deux cellules (i.e. 2D). Nous faisons l’hypothèse que les contraintes physiques à cette l’interface telles que la force et le mouvement relatif des molécules confinées aux surfaces modulent les propriétés de la liaison anticorps-antigène. Notre but est d’explorer les liens entre la mécanique de la liaison et la réponse cellulaire. Pour quantifier la cinétique 2D et la mécanique de ces interactions, nous avons effectué des mesures en utilisant la chambre à flux laminaire des deux anticorps à domaine unique (sdAbs) ciblant le récepteur CD16 exprimé dans la cellule Natural Killer (NK) et cinq sdAbs ciblant le marqueur tumoral HER-2 exprimé dans certains cancers. Nos résultats montrent des liaisons glissantes, idéales et pour la première fois, une liaison accrocheuse dans des interactions anticorps-antigène. Des expériences d’adhésion cellulaire montrent une corrélation entre la résistance à la force de la liaison accrocheuse et une meilleure adhésion des NK. Des sdAbs ont été sélectionnés pour constituer des anticorps bi-specifiques (bsAbs) capables de recruter des NK contre des cellules cancéreuses HER-2+. Ces bsAbs induisent une cytotoxicité supérieur a celle de l’anticorps de référence. Leur efficacité est modulée par la mécanique du coté antiCD16 du bsAbs en fonction de la nature de la cellule cancéreuse, suggérant un rôle de la force pour les faibles densités de HER-2. / Therapeutic antibodies have become a major treatment in cancer due in part to their ability to recruit immune cells onto tumours. They are selected on the basis of their affinity for their antigen in a three dimensions (3D) environment. However, in some major modes of action, antibodies do bind the antigen at the interface between immune cells and target cells. We hypothesize that the physical constraints of cell-cell interface (i.e. 2D), including force and relative motion of molecules confined at surfaces, modulate the antigen-antibody binding. Specifically, we aim at exploring the links between bond mechanics and cellular response. To quantify 2D kinetics and mechanics, we perform measurements using the laminar flow chamber of two Single Domains Antibodies (sdAbs) against the surface receptor CD16 expressed in Natural Killer (NK) cells and five sdAbs against the tumoral marker HER-2 expressed in some breast cancers. Our results show three different bond dissociation behaviour under force; slip, ideal and for the first time, a catch bond. Cell adhesion experiments over sdAb antiCD16 coated surfaces reveal a correlation between antibody resistance to force and a larger spreading of NK cells. Based on their force behaviour, some sdAbs were selected to be fused forming bi-specific antibodies (bsAbs) able to recruit NK cells toward HER-2+ cancer cells. All new bsAbs display a better efficacy in cytotoxicity than the reference therapeutic antibody. We show that their efficacy is modulated by the mechanical behaviour of the antiCD16 side, depending on the nature of the target cell line, which may hint to an effect of force dependence in the limit of low antigen coverage.