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Nous nous sommes intéressés au TMC-95A, un tripeptide cyclique naturel inhibant le protéasome humain d’une façon non-covalente à des concentrations de l’ordre du nanomolaire. Nous avons montré que des mimes linéaires du TMC-95A contenant un motif 3-hydroxyoxindolyl alanine conservent l’activité inhibitrice de ce dernier. Mon travail a porté sur la synthèse d’une bibliothèque de mimes linéaires du TMC-95A en partant des amino-acides protéogéniques et des dérivés de la 3-hydroxyoxindolyl alanine. Nous avons préparé des bras espaceurs hydrosolubles contenant des motifs éthylène glycol que nous avons couplés avec les têtes inhibitrices pour l’élaboration de nouveaux inhibiteurs bivalents capables de cibler simultanément deux des six sites catalytiques du protéasome constitutif et de l’immunoprotéasome.Au cours de la synthèse de la 3-hydroxyoxindolyl alanine, il a été observé qu’en milieu basique, ce résidu C-protégé se transpose lentement et d'une façon peu décrite dans la littérature en γ-lactame. Nous avons généralisé cette réaction à la synthèse de lactames de 5 à 12 chaînons par isomérisation d’oxindoles substitués en N-1 par un groupement électroattracteur et en C-3 par une chaine ω-aminée de longueur variable. Ces oxindoles ayant une chaîne aminée ont été obtenus à partir de deux familles différentes : des composés nitrés et des dérivés N-Boc protégés. L’aptitude des différentes molécules synthétisées à inhiber le protéasome et l’immunoprotéasome a été étudiée. / Nous nous sommes intéressés au TMC-95A, un tripeptide cyclique naturel inhibant le protéasome humain d'une façon non-covalente à des concentrations de l'ordre du nanomolaire. Nous avons montré que des mimes linéaires du TMC-95A contenant un motif 3-hydroxyoxindolyl alanine conservent l'activité inhibitrice de ce dernier. Mon travail a porté sur la synthèse d'une bibliothèque de mimes linéaires du TMC-95A en partant des amino-acides protéogéniques et des dérivés de la 3-hydroxyoxindolyl alanine. Nous avons préparé des bras espaceurs hydrosolubles contenant des motifs éthylène glycol que nous avons couplés avec les têtes inhibitrices pour l'élaboration de nouveaux inhibiteurs bivalents capables de cibler simultanément deux des six sites catalytiques du protéasome constitutif et de l'immunoprotéasome. Au cours de la synthèse de la 3-hydroxyoxindolyl alanine, il a été observé qu'en milieu basique, ce résidu C-protégé se transpose lentement et d'une façon peu décrite dans la littérature en γ-lactame. Nous avons généralisé cette réaction à la synthèse de lactames de 5 à 12 chaînons par isomérisation d'oxindoles substitués en N-1 par un groupement électroattracteur et en C-3 par une chaine ω-aminée de longueur variable. Ces oxindoles ayant une chaîne aminée ont été obtenus à partir de deux familles différentes : des composés nitrés et des dérivés N-Boc protégés. L'aptitude des différentes molécules synthétisées à inhiber le protéasome et l'immunoprotéasome a été étudiée.

Le récepteur au goût sucré est un hétérodimère composé des sous-unités T1R2 et T1R3, alors que les sous-unités T1R1 et T1R3 s’assemblent pour composer le récepteur au goût umami. Chacune de ces sous-unités appartient à la classe C des récepteurs couplés aux protéines G (RCPG). Les membres de cette famille partagent une architecture commune, constituée d’un domaine N-terminal (DNT) qui est relié à un domaine transmembranaire par une région riche en cystéines. Le DNT est constitué de deux lobes qui forment le site orthostérique, qui est le site de liaison des principaux agonistes. Il a été montré que les DNT de T1R1 (DNT-T1R1) est capable de lier le L-glutamate alors que le DNT de T1R2 (DNT-T1R2) est capable de lier les sucres naturels et certains édulcorants (Zhang et al., 2008 ; Nie et al., 2005). Cependant les mécanismes moléculaires de détections des molécules sapides au niveau de ces domaines restent encore largement méconnus. Lors de cette étude, nous avons exprimé les DNT-T1R1, DNT-T1R2 humains et le DNT-T1R1 de souris à l’aide de la bactérie Escherichia coli, sous forme de protéines insolubles, appelés corps d’inclusion (CI). Les CI ont été purifiés puis solubilisés en utilisant un agent chaotrope. Ensuite, les protéines ont été repliées in vitro puis caractérisées par différentes approches parmi lesquelles l’électrophorèse, la filtration sur gel, la fluorescence et le dichroïsme circulaire. Leur fonctionnalité a ensuite été vérifiée par microcalorimétrie de titration isotherme. Nous avons montré que les protéines sont fonctionnelles et présentent des affinités en accord avec les données sensorielles. Les grandes quantités de protéines produites permettront de futures études structurales par cristallographie. Parallèlement à ce travail, nous avons étudié un polypeptide végétal, inhibiteur des goûts sucré et umami chez les rongeurs, appelé gurmarine. Afin de pouvoir étudier son interaction avec les DNT des récepteurs gustatifs de souris, nous avons mis au point l’expression et la purification de gurmarine recombinante produite en levure Pichia pastoris. Ce système d’expression a permis la production de quantités importantes de gurmarine dont les caractéristiques structurales ont été vérifiées par dichroïsme circulaire et résonnance magnétique nucléaire. Nous avons aussi, par mutagénèse dirigée, modifié différents acides aminés décrits comme étant potentiellement impliqués dans l’interaction avec les récepteurs aux goûts sucré et umami. En collaboration, l’activité de la gurmarine a été confirmée à l’aide d’un test cellulaire basé sur l’expression fonctionnelle du récepteur de rat T1R2/T1R3. A l’aide de différentes combinaisons des sous-unités humaines et de rongeurs, nous avons montré que l’inhibition par la gurmarine nécessitait la présence de la sous-unité T1R3 de rat. / The sweet taste receptor is a heterodimer composed of two subunits called T1R2 and T1R3 whereas the T1R1 and the T1R3 subunits form a heterodimeric receptor for umami taste (the savory taste of monosodium glutamate). Each subunit belongs to the class C of G protein-coupled receptors (GPCRs) and is constituted by a large extracellular N-terminal domain (NTD) linked to the transmembrane domain by a cysteine-rich region. The NTD is composed of two lobes separated by a cleft in which ligands bind. T1R1- and T1R2-NTDs are able to bind sweeteners and umami compounds respectively and undergo ligand-dependent conformational changes (Zhang et al., 2008; Nie et al., 2005). However, the relative contribution of the two subunits to the heterodimeric receptor function remains largely unknown. To study the binding specificity of each subunit, a large amount of purified NTDs is suitable for biochemical and structural studies. To accomplish this goal, we expressed T1R1- and T1R2-NTD in high level in Escherichia coli as insoluble aggregated proteins (inclusion bodies). The proteins were solubilized and in vitro refolded using suitable buffer and additives. The soluble proteins were then purified and characterized using electrophoresis, gel filtration, fluorescence spectroscopy and circular dichroism. The functionality of T1R1- and T1R2-NTD was measured the using isothermal microcalorimetry. Our data showed that the proteins are properly refolded and able to bind sweet or umami compounds with physiological relevant affinities. In summary, our expression system will allow large-scale production of active T1R1- and T1R2-NTD suitable for structural and functional studies. In addition to this work, we have studied a 35 residue polypeptide named gurmarin, well known to selectively inhibit responses to sweet substances without affecting responses to other basic taste stimuli, such as NaCl, HCl, and quinine in rodents. To further understand the structural basis of gurmarin recognition by T1R2-T1R3 receptor, we developed for the first time the heterologous expression of gurmarin using the methylotrophic yeast Pichia pastoris. This system allowed the expression of large quantities of recombinant gurmarin. The structural properties of gurmarin were checked by circular dichroism and nuclear magnetic resonance. We generated six mutants with single amino acids substitutions in the putative site of interaction between gurmarin and the rodent sweet taste receptor, using site-directed mutagenesis. In collaboration, the biological activity of was confirmed using a cell-based assay based on expression of rat T1R2-T1R3. Thanks to various combinations of human and rat T1R2-T1R3 chimeras, we showed that NTD of rat T1R3 is the major determinant of gurmarin’s inhibition.

Protein kinases are important regulatory enzymes in signal transduction and in cell regulation. Understanding inhibition mechanisms of kinases is important for the further development of new therapies for cancer and inflammatory diseases. I have developed a statistical approach based on the Mantel test to find the relationship between the shapes of ATP binding sites and their affinities for inhibitors. My shape-based dendrogram shows clustering of the kinases based on similarity in shape. I investigate the pocket in terms of conservation of surrounding amino acids and atoms in order to identify the key determinants of ligand binding. I find that the most conserved regions are the main chain atoms in the hinge region and I show that the tetrahydropyran ring of staurosporine causes induced-fit of the glycine rich loop. I apply multiple linear regression to select distances measured between the distinctive parts of residues which correlate with the binding constants. This method allows me to understand the importance of the size of the gatekeeper residue and the closure between the first glycine of the GXGXXG motif and the aspartate of the DFG loop, which act together to promote tight binding to staurosporine. I also find that the greater the number of hydrogen bonds made by the kinase around the methylamine group of staurosporine, the tighter the binding to staurosporine. The website I have developed allows a better understanding of cross reactivity and may be useful for narrowing down the options for a synthetic strategy to design kinase inhibitors.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Fluoxetine (FLX), the active ingredient of Prozac™, is a member of the selective serotonin reuptake inhibitor (SSRI) class of anti-depressant drugs and is present in aquatic environments worldwide. Previous studies reported that FLX is an endocrine disruptor in fish, bioconcentrating in tissues including the brain. Evidence implicates that serotonin influences the activity of the hypothalamo-pituitary-interrenal (HPI) stress axis, thus exposure to FLX may disrupt the teleost stress response. This study examined in vitro cortisol production in rainbow trout (Oncorhynchus mykiss) head kidney/interrenal cells exposed to FLX and 14C-pregnenolone metabolism in head kidney microsome preparations of FLX-exposed trout. Results indicated that cells exposed in vitro to increasing concentrations of FLX had lower cortisol production and cell viability (versus control) and microsomes isolated from trout exposed to 54 μg/L FLX had higher pregnenolone metabolism versus those of control and low FLX-exposed (0.54 μg/L) trout.

Skeletal muscle atrophy is a debilitating condition caused by pathological conditions including cancer cachexia, disuse and denervation. Disuse atrophy is characterized by reduction in fiber size, fiber-type change and induction of markers of atrophy such as MuRF1 and Fn14. Recent studies have focused on understanding the fundamental role of signalling pathways and the proteolytic system in response to muscle atrophy. Unfortunately the exact mechanisms behind atrophy remain poorly understood. I recently demonstrated that cIAP1 and/or cIAP2 proteins are critical regulators of NF-kB activation, which has been shown to be involved in skeletal muscle atrophy. Here, I used genetic and pharmacological means to investigate the role of cIAP1 in a denervation-induced skeletal muscle atrophy model. Interestingly, I found that upon denervation loss of cIAP1 rescues muscle fiber size, prevents fiber-type changing and inhibits the expression of MuRF1 and Fn14. Moreover, treatment of mice with Smac mimetic compounds (SMC), a novel class of small molecule IAP antagonists, showed successful knockdown of cIAP1 in muscle and protects against denervation-induced muscle atrophy. Taken together, these data reveal that cIAP1 is both a novel mediator of skeletal muscle atrophy and an important therapeutic target.

Trichostatin A (TSA) is a histone deacetylase inhibitor with beneficial effects in spinal muscular atrophy mouse models that carry the human SMN2 transgene. Whether TSA specifically targets the upregulation of the SMN2 gene or whether other genes respond to TSA and in turn provide neuroprotection in SMA mice is unclear. We have taken advantage of the Smn2B/- mouse model that does not harbor the human SMN2 transgene, to test the hypothesis that TSA has its beneficial effects through a non-Smn mediated pathway. Daily intraperitoneal injection of TSA from postnatal day 12 to 25 was performed in the Smn2B/- mice and littermate controls. Previous work from our laboratory demonstrated that treatment with TSA increased the median lifespan of Smn2B/- mice from twenty days to eight weeks. As well, there was a significant attenuation of weight loss and improved motor behavior. Pen test and righting reflex both showed significant improvement, and motor neurons in the spinal cord of Smn2B/-mice were protected from degeneration. Both the size and maturity of neuromuscular junctions were significantly improved in TSA treated Smn2B/- mice. Here, we have shown that TSA treatment does not increase the levels of Smn protein in mouse embryonic fibroblasts or myoblasts obtained from the Smn2B/- mice. Further, qPCR analysis revealed no changes in the level of Smn transcripts in the brain or spinal cord of TSA-treated SMA mice. Similarly, western blot analysis revealed no significant increase in Smn protein levels in the brain, spinal cord, hind limb muscle, heart muscle, or the liver of TSA treated Smn2B/- mice. However, TSA has beneficial effects in the muscles of Smn2B/- mice and improves motor behavior and myofiber size. TSA improves muscle development by enhancing the activity of myogenic regulatory factors independent of the Smn gene. The beneficial effect of TSA is therefore likely through an Smn-independent manner. Identification of these protective pathways will be of therapeutic value for the treatment of SMA.

Les glycosyltransférases sont responsables de la biosynthèse d’oligosaccharides, de polysaccharides et de glycoconjugués. Etant donné le nombre croissant de processus biologiques reliés aux saccharides, il existe un grand intérêt pour la compréhension des rôles biologiques des ces saccharides et étudier leurs applications thérapeutiques potentielles. Ce projet concerne la conception, la synthèse, l’évaluationbiologique et l’analyse structurale de deux nouveaux types d’inhibiteurs de glycosyltransférases analogues du substrat donneur naturel. L’analogie repose sur l’incorporation d’une unité pyridine ou amino-acide neutre comme mime du motif pyrophosphate afin de fournir des substrats capables de pénétrer les cellules pour des applications potentielles in cellulo ou in vivo. Les synthèses d’inhibiteurs neutres de GTs ont été réalisées en utilisant une combinaison de réactions de conjugaison créant une liaison O-glycosidique, amide ou triazole. Au total, 26 inhibiteurs neutres de GTs ont ainsi été synthétisés. L’évaluation de l’inhibition pour cinq galactosyltransférases et une GlcNAc-transférase(OGT) a révélé des inhibitions modestes de l’ordre du micromolaire. La co-cristallisation des meilleurs inhibiteurs avec l’une de ces galactosyltransférases a démontré que le motif pyridine neutre chélate le cation manganèse impliqué dans le site catalytique de l’enzyme. Cependant, le motif galactose est orienté vers l’extérieur du site catalytique et loin de la position initiale du substart naturel (UDP-Gal) etindique donc un nouveau mode de liaison. Le concept d’inhibiteur neutre a aussi été examiné sur un système modèle de membrane pour leur perméation membranaire. / Glycosyltransferase is an important class of enzyme in living organisms responsible for the biosynthesis of oligosaccharides, polysaccharides and glycoconjugates. As more and more carbohydrate related biological processes are elucidated, there is great interest to define the biological roles of a given carbohydrate and examine its potential therapeutic applications. The present study reports the design, synthesis, biological evaluation and structural analysis of two novel types of glycosyltransferase donor substrate analogues. The design rationale is to make analogues of sugar nucleotide diphosphate substrates, but incorporating a ‘neutral’ pyridine or amino-acid moiety as thepyrophosphate surrogate in order to provide cell permeable substrates for potential in cellulo or in vivo applications. The syntheses of “neutral” GTs inhibitors were performed using a combination of conjugations through O-glycoside bond, amide bond or triazole functionalities. A total number of 26 “neutral” GT inhibitors were prepared. The evaluation of inhibition towards five galactosyltransferasesand one GlcNAc-transferase (OGT) revealed moderate inhibitions in the micromolar range. More interestingly, co-crystallyzation could be achieved for the most potent compounds in complex with a glycosyltransferase. The designed ‘neutral’ pyridine linker could chelate the manganese cation involved in the enzyme catalytic site. Whereas the sugar head-group was oriented away from the position found inthe related complex with natural substrate (UDP-Gal) indicating a new binding mode. The concept of ‘neutral’ inhibitor was examined by an artificial cell membrane penetration test.

Under normal conditions, cathodically protected mild steel in seawater is protected by a precipitated film of calcium carbonate and magnesium hydroxide, the so-called calcareous film. This study has attempted to investigate the dynamics of calcareous deposit formation during cathodic protection and the composition of calcareous deposits formed under different applied current densities, and also the role played by the initial current density in forming a good quality calcareous deposit. In addition, an under protection situation can occur where current demand values are under estimated, or where structures are approaching the end of their design lives. In these conditions, a calcareous film might well occur but complete protection is probably not possible. These situations have also been studied. At low insufficient current densities where steel corrosion is still occurring, a clear correlation exists between the iron containing corrosion product and the overlaying magnesium hydroxide layer. Such effects have also been investigated using pH titration analysis, where the effect of co-precipitation of the iron and magnesium oxides/hydroxides has been shown. At higher current densities a layered precipitate has been shown to occur consisting of an inner magnesium containing layer and an outer calcium containing layer. At obvious overprotection current densities, the mechanical stresses involved in hydrogen evolution are assumed to give rise to film cracking. To augment and compliment the study on calcareous calcium/magnesium films formed during cathodic protection, a calcium-magnesium containing pigment has also been investigated in aqueous solutions at open circuit as a possible corrosion inhibitor. Another study looked at the same inhibitor in conjunction with a sacrificial zinc anode. Very effective inhibition has been shown with the film containing not only magnesium, calcium and phosphorous but also zinc. In all the investigations electrochemical methods have been used together with various surface analytical techniques.