La MAPK p38α est une protéine clé de l’inflammation, également impliquée dans de nombreux processus liés au cancer. Les petites molécules chimiques inhibitrices de p38α bloquent son activité kinase par un mécanisme de compétition à l’ATP. En raison de la grande conservation du domaine kinase, la spécificité de la majorité de ces inhibiteurs n’est pas restreinte à la p38α et des effets « off-targets » ont été rapportés. Dans ce contexte, le projet de ma thèse a porté sur l’utilisation de fragments d’anticorps au format scFv comme nouvel outil de ciblage pharmacologique afin de définir une/des voie(s) alternative(s) d’inhibition de la p38α. Les fragments d’anticorps lient un motif antigénique avec une affinité et une spécificité élevées, tout comme les immunoglobulines classiques. Leur expression intracellulaire permet également le ciblage de protéines cytoplasmiques et l’étude de leurs fonctions dans des processus physiologiques et pathologiques. Nous avons sélectionné par phage display, à partir d’une banque de fragments d’anticorps, 5 scFv spécifiques de la MAPK p38α. Alors que tous ces scFv empêchent l’activation par phosphorylation de la p38α par MKK6, l’un d’entre eux agit directement sur l’enzyme pour inhiber totalement son activité kinase in vitro. Ce scFv possède un site de liaison et un mécanisme d’inhibition distincts des inhibiteurs pharmacologiques déjà décrits : bien qu’il ne cible pas le domaine kinase et n’empêche pas la fixation de l’ATP, le scFv se comporte comme un inhibiteur compétitif de l’hydrolyse de l’ATP. Ces résultats suggèrent un effet allostérique du scFv sur l’activité de la p38α et permettent de le caractériser comme un inhibiteur compétitif non conventionnel. La détermination de son épitope d‘interaction ainsi que la confirmation de sa fonctionnalité une fois exprimé dans le cytosol des cellules nous permettra de définir une voie alternative d'inhibition de la p38α et de valider notre approche de ciblage par l’utilisation de fragments d’anticorps.Ces données ouvrent de nouvelles perspectives de design d’inhibiteurs chimiques de la p38α de meilleure spécificité que ceux actuellement disponibles. / MAPK p38α is a key protein in inflammation, but is also involved in many cancer-related processes. All the currently described chemical inhibitors of p38α inhibit its kinase activity by an ATP-competitive mechanism. Because of the high conservation of the ATP-binding pocket, the majority of these inhibitors are not specific to p38α and off-target effects have been reported. To identify alternative approaches to inhibit p38α MAPK, my thesis project focused on the use of scFv antibody fragments as a new highly specific pharmacological tool.Antibody fragments bind to an antigen with high affinity and specificity like conventional immunoglobulins. Their intracellular expression also allows to target cytoplasmic proteins and study the target functions in physiological and pathological processes. Using a naïve library of antibody fragments, we have selected by phage display five scFv specific of MAPK p38α isoform. While all these scFv inhibit the activation of p38α by MMK6, one of them also completely inhibits its kinase activity in vitro. This scFv has a binding site and a mechanism of inhibition distinct from the pharmacological inhibitors currently described: although it does not target the ATP-binding pocket and does not prevent ATP binding, it behaves like a competitive inhibitor of ATP hydrolysis. These results suggest an allosteric effect of this scFv on p38α activity and allow to characterize it as an unconventional competitive inhibitor. The determination of its epitope as well as the confirmation of its inhibitory activity once expressed in the cell cytosol will allow us to propose an alternative approach to target p38α function using antibody fragments.These data open up new perspectives for the design of more specific p38α chemical inhibitors than those currently available.

Submitted by FERNANDA DA SILVA VON PORSTER (fdsvporster@univates.br) on 2016-09-22T19:02:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Approved for entry into archive by Ana Paula Lisboa Monteiro (monteiro@univates.br) on 2016-09-29T19:20:07Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) / Made available in DSpace on 2016-09-29T19:20:08Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) 2016JulianoLeipelt.pdf: 2508061 bytes, checksum: 100e43c73eddacc8441a11de473e260c (MD5) Previous issue date: 2016-09 / O câncer é apontado como a segunda maior causa de morte em todo o mundo, com previsão de em breve ser tornar a primeira. O câncer de próstata está entre os 5 tipos de câncer mais diagnosticados em homens, sendo que o câncer hepático está em segundo lugar em taxa de mortalidade entre homens e mulheres. Indícios apontam para uma ativação das vias da inflamação associados a uma inibição das vias de morte celular no processo de carcinogênese. A regulação destas vias torna-se alvo importante e complementar no controle do câncer, sendo estimulada a busca de biomoléculas com este potencial. As plantas são importante fonte de descoberta de novas biomoléculas com ampla utilização para o tratamento de diversas patologias. A família Myrtaceae possui diversas espécies que são apontadas como fortes candidatos em potencial nesta busca, incluindo as do gênero Myrciaria. A espécie Myrciaria plinioides não possui estudos referentes suas propriedades terapêuticas ou a atuação em vias de sinalização envolvidas na inflamação ou na carcinogênese. Neste contexto, este estudo teve por objetivo avaliar a atividade do extrato etanólico de M. plinioides em células de carcinoma hepatocelular (HepG2) e próstata (LNCaP) , através da análise de expressão dos marcadores p38-α, pp38-α, NF-κB e caspase-3, envolvidos na carcinogênese, e o efeito sobre a viabilidade celular através do método de MTT. A viabilidade das células foi alterada significativamente, em ambas as linhagens celulares quando tratadas com o extrato etanólico. A análise da expressão proteica demonstra significativa inibição da expressão de p38-α e caspase-3 nas células LNCaP, quando tratadas com extrato etanólico de M. plinioides seguido de LPS. Em células HepG2, somente houve alteração na expressão da caspase-3 na concentração de 200 μg/mL, com ou sem adição de LPS após tratamento com extrato. Os resultados deste estudo demonstraram redução da viabilidade celular nas duas linhagens tumorais, expressão diferenciada de proteínas envolvidas em apoptose, o que leva a indícios da ativação de mecanismos distintos pelo extrato em cada tipo celular. Estudos futuros para averiguar o mecanismo celular e a indução de morte em células tumorais de câncer de próstata e de fígado podem contribuir para a identificação e elucidação de novas biomoléculas com potencial antitumoral. / Cancer is touted as the second leading cause of death worldwide, forecast to soon be making the first. Prostate cancer is among the five most cancers diagnosed in men, and liver cancer is second in mortality between men and women. Evidence points to the activation of pathways of inflammation associated with an inhibition of cell death pathways in carcinogenesis. The regulation of these pathways becomes important and complementary target in cancer control, and stimulated the search for biomolecules with this potential. The plants are important source of discovery of new biomolecules with wide use for the treatment of various diseases. The Myrtaceae family has many species that are identified as potential candidates strong in this search, including the Myrciaria genre. The species Myrciaria plinioides not have studies on its therapeutic properties or performance in signaling pathways involved in inflammation or carcinogenesis. In this context, this study aimed to evaluate the activity of the ethanol extract of M. plinioides in hepatocellular carcinoma cells (HepG2) and prostate (LNCaP) by expression analysis of p38-α markers, PP38-α, NF-kB and caspase-3, involved in carcinogenesis, and the effect on cell viability by the MTT method. The viability of cells was significantly altered in both cell lines when treated with ethanolic extract. Protein expression analysis demonstrates significant inhibition of p38-α expression and caspase-3 in LNCaP cells, when treated with ethanolic extract of M. plinioides followed by LPS. In HepG2 cells there was only a change in the expression of caspase-3 at a concentration of 200 / ml, with or without addition of LPS after treatment with extract. The results showed reduction of cell viability in both tumor lines, differential expression of proteins involved in apoptosis, leading to evidence of activation by distinct mechanisms in each extract cell type. Further studies to investigate the cellular mechanism, and induction of death in tumor cells of prostate and liver cancer may contribute to the identification and elucidation of new biomolecules with antitumor potential.

It is widely known that early estimates about the binding properties of drug candidates are important in the drug discovery process. Surface plasmon resonance (SPR) biosensors have become a standard tool for characterizing interactions between a great variety of biomolecules and it offers a unique opportunity to study binding activity. The aim of this project was to develop a SPR based assay for pre-screening of low molecular weight (LMW) drug compounds, to enable filtering away disturbing compounds when interacting with drugs. The interaction between 47 LMW compounds and biological ligands were investigated using the instrument BiacoreTM, which is based on SPR-technology.