La dépression est une maladie psychiatrique qui atteint environ 20% de la population. En 2006 notre équipe a montré l’implication du canal potassique TREK-1 dans cette pathologie, et, en 2010, elle a identifié un bloqueur de TREK-1, la spadine, ayant des propriétés antidépressives. La spadine a la même efficacité après quatre jours de traitement que les antidépresseurs classiques qui en nécessitent vingt-et-un. Lors de mon doctorat, nous avons démontré la spécificité de la spadine pour les canaux TREK-1 et l’absence d’effet secondaire notamment au niveau cardiaque et sur les fonctions où l’activation du canal TREK-1 a des effets bénéfiques (ischémie, épilepsie, douleur). Nous avons pour cela utilisé des techniques d’électrophysiologiques et différents tests comportementaux adaptés à ces différentes pathologies. J’ai également démontré les effets antidépressifs de la spadine sur deux modèles animaux, un modèle génétique, les souris Rouen, et un modèle induit par un traitement à la coticostérone. La dernière partie de ma thèse a porté sur l’identification d’analogues de la spadine ayant une meilleure affinité et une meilleure efficacité in vivo, et ne présentant pas d’effet secondaire. Deux peptides synthétisés par la technique de retro-inverso ont présenté ces propriétés. Parallèlement, nous avons également recherché, en collaboration avec la société MedinCell, une formulation de polymère permettant la libération constante et prolongée de l’analogue sélectionné. Cette formulation a pour but de résoudre les problèmes engendrés par le non-respect de la prise de médicaments par les patients en administrant en une seule injection le traitement pour trois voire quatre semaines. / Depression is a devastating psychiatric disorder which affects about 20% of the population. In 2006 our team demonstrated the involvement of TREK-1, a potassium channel, in this pathology, and that its inhibition has led to a depression’s resistant phenotype. The search of TREK-1 inhibitors, a potential antidepressant, has led to the discovery of spadin. Spadin has, after only a four day treatment, similar efficacy than classical antidepressants which require about three weeks of treatment to produce their therapeutic effects. My work was firstly focused on the potential side effects of spadin. Indeed, TREK- 1’s activation has beneficial effects in many pathophysiologies (ischemia, epilepsy, pain). Its inhibition by spadin could generate significant adverse effects. The use of animal models has allowed us to confirm that spadin has no side effects related to TREK-1 channel’s inhibition. The specificity of spadin has been demonstrated since it has no effect on other potassium channels belonging to the K2P family. My work was also to study the effects of spadin in two mice models of depression, a genetic model and an induced model. In both cases, spadin shows a specific antidepressant effect in different behavioral tests of depression. The last part of my work was focused on the development of spadin’s analogs in order to improve the affinity and in vivo efficiency. We also developed, in collaboration with the MedinCell society, a polymer formulation for the constant and prolonged release of the selected analog. This formulation will be administered as a single injection treatment for three to four weeks, solving the problems caused by non respected medication by patients.

The intrinsic plasticity of biological systems provides opportunities for rational design of selective and potent ligands. Increasingly, computational methods are being applied to predict biomolecular flexibility. However, the motions involved in these processes can be large and occur on time scales generally difficult to achieve with standard simulation methods. In order to overcome the intrinsic limitations of classical molecular dynamics, this Ph.D. project focuses on the application of advanced sampling computational techniques to capture the plasticity of diverse biological systems. The first of these applications involved the evaluation of the secondary structure of the N-terminal portion of p53 and its inverse, reverse and retro-inverso sequences by using replica exchange molecular dynamics simulations in implicit solvent. In this study, we also evaluated the effects of reversal of sequence and stereochemistry in mimicking an inhibitory pharmacophoric conformation. The results showed how the ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). Moreover, the structural information obtained from simulations showed good agreement with NMR and circular dichroism studies, confirming the validity of the combination of replica exchange molecular dynamics with the ff99SB force field and Generalized Born solvent model for computational modelling of D-peptide conformations.In a second work, we explored conformations of the DFG motif of the p38α mitogen-activated protein (MAP) kinase. To achieve this, we employed an advanced sampling simulation method that has been developed in-house, called swarm-enhanced sampling molecular dynamics (sesMD). In contrast to multiple independent MD simulations, swarm-coupled sesMD trajectories were able to sample a wide range of DFG conformations, some of which map onto existing crystal structures. Simulated structures intermediate between DFG-in and DFG-out conformations were predicted to have druggable pockets of interest for structure-based ligand design. Overall, sesMD shows promise as a useful tool for enhanced sampling of complex conformational landscapes. Finally, we used microsecond MD simulations to evaluate the molecular plasticity of R-spondins, a class of proteins involved in the activation of the Wnt pathway. The unbound R-spondin 1 is characterised by a closed conformation, while, when complexed to proteins LGR and RNF43/ZNRF3, assumes an open and more extended arrangement. This is true also for R-spondin 2, in both its unbound or bound forms. From our simulation, we find that the closed R-spondin 1 conformation is stable, whilst, R-spondin 1 and 2 from their open conformation explore several intermediate structures. In addition, we evaluated the druggability of a potential binding site located at the interface between the second and the third β-hairpin moiety of the first furin domain. The computational screening with small molecular fragments provided interesting insights about the druggability and the pharmacophoric features of the potential binding pockets identified, outlining promising future perspectives of structure-based design of Wnt pathway inhibitors.

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