Inhibiteur sélectif de la recapture de la sérotonine

Les inhibiteurs sélectifs de la recapture de la sérotonine (ISRS ; SSRI (Selective Serotonin Reuptake Inhibitor) en anglais) sont une classe médicamenteuse de psychotropes, de type antidépresseurs. Ils opèrent dans le cerveau, notamment en augmentant le taux d'un neurotransmetteur appelé sérotonine (5-hydroxytryptamine ou 5-HT). Ils agissent en inhibant la recapture de la sérotonine au niveau de la synapse. Les ISRS sont fréquemment prescrits, outre leur indication principale qu'est la dépression, dans les cas d'anxiété et de trouble obsessionnel compulsif et dans les douleurs neuropathiques réfractaires (duloxétine), en réduisant la charge émotionnelle, en augmentant le tonus (effet booster) et en créant une désinhibition potentiellement dangereuse (virage maniaque, bipolarité iatrogène au niveau du gyrus cingulaire). Ils ont aussi un effet négatif sur la libido (diminution ou absence de désir sexuel) ; cet effet indésirable permet de retarder l'orgasme chez les hommes souffrant d'

Thrombosis efficacy induced by photodynamic therapy using different photosensitizers

Bernadette Pegaz-Toquet

Various human pathologies, such as cancers or, notably, exudative age-related macular degeneration (AMD), are characterized by an abnormal vascular proliferation (angiogenesis), which in the case of this type of AMD, can rapidly lead to blindness if no countermeasures are taken. In the past few years, it has become possible to stop - or at least to notably slow down - the evolution of AMD, by applying treatments based on photodynamic therapy (PDT), whereby, through the action of a molecule acting as photosensitizer (PS) which is targeted preferentially to the choroidal neovessels (CNV), the effects of this abnormal angiogenesis are stopped by provoking the closure of the neovessels. In particular, the PDT group at EPF-Lausanne has, in the past, already tested on the model of the chick embryo chorioallantoic membrane (known as the CAM model) several PSs of potential interest as substitute to the product currently considered as standard (Visudyne® , itself co-developed in this group) for the treatment of the CNV associated with AMD. The aim of this thesis was to study new molecules and/or formulations, potentially suitable as substitutes to the current photodynamic treatment of AMD, while diminishing its negative side-effects: general phototoxicity due to diffusion of the product out of the vasculature and further lack of selectivity leading to (undesirable) partial occlusion of the normal vasculature. In order to enable extrapolation and comparison of the results obtained, we have focused our attention on vascular effects only and administered the tested PSs under essentially similar conditions (formulation in DMSO; same delivery system through biodegradable nanoparticles built with the same polymer). In addition, the same animal model (the CAM model) was used throughout the entire study, the formulations being administered by intravenous injection. This model, importantly, allows for an easy and real-time semi-quantitative visualisation and recording of the vascular effects of a PS induced during a PDT session. In the first part of this work, we demonstrate that the tetra-phenylporphyrin molecule, when formulated in nanoparticles based on poly(D, L - lactic acid, of well defined size (ca. 200 nm or less) is a very interesting PS-candidate potentially suitable as a replacement of the currently used one. We show that inclusion of this PS in nanoparticles notably reduces the PDT's general phototoxicity. We believe therefore that this PS would deserve more thorough pre-clinical studies, on an animal model closer to the human case (for example, Ryan's model). The second part of this thesis aimed at elaborating a strategy allowing, in due time, for an improved selectivity of occlusion of the neovasculature, said strategy aiming at an active targeting of neovessels. Recently, a number of possible biochemical targets have been identified, including the so-called ανβ3 receptors, which are highly expressed at the level of the neovessels' endothelial cells. Our approach consisted in coupling a fluorescent molecule (fluorescein, easily visualized by epi-microscopy) to a protein (a kistrin) which contains the RGD peptide sequence, known for its affinity for the ανβ3 receptors. We have thus been able to demonstrate that the kistrin was effectively linked to these receptors and, therefore, that active targeting of the neovessels had indeed taken place. A complete proof of this concept by realizing the coupling of this kistrin with a PS molecule will be the next step in this research.

EPFL2007

Improving Bicyclic Peptide Phage Display and Development of Sortase A Inhibitors

Inmaculada Rentero Rebollo

Bicyclic peptide ligands are promising molecules for the development of new therapeutics. They combine advantages from large protein therapeutics and small molecule drugs. Large combinatorial libraries of bicyclic peptides can be generated and screened by phage display using a recently developed strategy. Potent and selective bicyclic peptide inhibitors against several therapeutic targets have already been developed and their therapeutic potential is currently being evaluated in animal models. One aim of my thesis was the exploration of ring size diversity in bicyclic peptides. I generated a set of libraries of the format Cys-(Xaa)m-Cys-(Xaa)n-Cys, where 'm' and 'n' = 3, 4, 5 or 6, and performed affinity selections against the serine protease urokinase-type plasminogen activator. Bicyclic peptide inhibitors from virtually all ring size combinations were isolated, suggesting that many peptide formats can be accommodated in the active site of this enzyme. Moreover, they showed a large variety of consensus sequences and several of the identified consensus sequences were exclusively found in bicyclic peptides having specific ring size combinations. Having available multiple leads isolated from such bicyclic peptide libraries with variable ring sizes could be a great asset for the generation of high affinity binders. Additionally, other targets may have preferences for specific peptide constraints and the availability of these libraries increases the chances to isolate high affinity binders to any desired target. A second goal of my thesis was to apply high throughput sequencing technologies to phage display selections of bicyclic peptides, in order to identify a larger number of specific target-binding sequences and motifs. I developed a procedure to efficiently compare the sequences of large numbers of phage-selected peptides to identify target-binding peptide motifs based on abundance and sequence similarity. Applying this approach to phage isolated in selections against five different protein targets, I was able to identify rare target-binding peptide motifs and could more precisely define groups and sub-groups of consensus sequences. This information is valuable to choose peptide leads for drug development and facilitates the identification of epitopes. Moreover, binding motifs could be identified after a single round of phage panning. The final aim of my thesis was to discover bicyclic peptides that could be used as new antibiotics. Towards this end, I combined the newly generated variable ring size libraries and high-throughput sequencing procedures. I focused on the development of inhibitors of Staphylococcus aureus sortase A, an antivirulence target for which no potent and specific inhibitors have been reported. For the isolation of bicyclic peptide inhibitors to this target, the ring size diversity of the libraries turned out to be key. Inhibitors all shared the same motif in a loop of 5 residues. Further characterization of their effects on S. aureus showed that they could inhibit sortase-mediated incorporation of external substrates on the staphylococcal cell wall. However, they were not sufficiently potent to compete with the native substrates of the enzyme. More potent inhibitors are needed to effectively inhibit sortase A on S. aureus cells, and the bicyclic peptide inhibitors isolated constitute promising leads for the development of future antisortase therapeutics.

EPFL2014

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport

Andrea Volterra

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.