Le déséquilibre HAT/HDAC aurait une influence sur le développement de certains cancers ainsi que dans l’addiction à l’alcool ou à la cocaïne. En inhibant les histones désacétylases, le taux d'acétylation de la chromatine augmente ce qui permet l’accès aux facteurs de transcription et l'expression des gènes. Aujourd'hui, il existe de nombreux inhibiteurs d'HDAC de structures diverses, mais ils ne sont pas spécifiques et présentent des effets secondaires importants. Les inhibiteurs d'HDAC comme le butyrate de sodium ou le MS-275 ont montré une modification de la dépendance à l'alcool chez le rat. MS-275 inhibe principalement la classe I de HDAC et conformément à ces observations nous nous intéressons aux inhibiteurs les plus sélectifs de la classe I tels que le Largazole thiol et le RedFK228. Notre but est de synthétiser de nouveaux cyclodepsipeptides analogues afin d'obtenir un inhibiteur sélectif de la classe I. Les HDAC de la classe I sont Zn-dépendants, ces analogues auront un groupement sulfonylhydrazide ayant une bonne affinité pour l’ion Zn2+ (ZBG). Il sera relié au cyclodepsipeptide par un bras espaceur dont la longueur sera adaptée (n = 2, 3). Une autre pharmacomodulation concerne l'incorporation d’hétérocycles différents (oxazole, thiazole et pyridine). Les inhibitions de ces composés ont pu être testées sur HDAC1, HDAC3 et HDAC6. Un composé a une spécificité pour HDAC3 et un autre a une spécificité pour HDAC1. Les tests sur des rats "binger" permettent de penser que HDAC1 est impliqué dans ce model de consommation et non HDAC3. / The imbalance HAT/HDAC would influence the development of cancers and alcohol or cocaine addiction. HDAC inhibition allows increase of both acetylation rate and gene expression. Today, there are many structurally diverse potent, but non-specific HDAC inhibitors displaying important side-effects. HDAC inhibitors such as sodium butyrate or MS-275 have been shown to alter the alcohol dependence in the rat. MS-275 inhibits mainly class I of HDAC and in line with these observations we are interested in more selective class I inhibitors such as Largazole thiol and RedFK228. Our purpose is to synthesize new cyclodepsipeptides analogues in order to obtain selective class I inhibitor. HDAC class I is a Zn-dependent enzyme and our target molecules have sulfonylhydrazide function as efficient Zinc binding group (ZBG). Additional pharmacomodulations concern the incorporation of different heterocycles (oxazole, thiazole, pyridine) and varying linker lengths (n = 2, 3). Inhibitions of these compounds have been tested on HDAC1, HDAC3 and HDAC6. A compound has specificity for HDAC3 and another has specificity for HDAC1. Tests on rats "binger" suggest that HDAC1 is involved in this model of consumption and not HDAC3.

Class of 2014 Abstract / Specific Aims: To identify reasons that an angiotensin converting enzyme inhibitor (ACEi) would not be indicated in diabetic patients with repeated clinical alerts to add ACEi therapy for preservation of renal function and/or hypertension. In addition, to identify if these repeated clinical alerts to add ACEi therapy are appropriate. Methods: Eligible patient charts were reviewed by researchers using a data dictionary to complete a standardized spreadsheet with patient demographic information (age, gender, and location), type of diabetes mellitus, evidence indicative of comorbid hypertension, action taken by pharmacist in response to clinical alert (letter sent to patient and letter sent to prescriber), and rationale of that action. This data, along with SOAP notes of patient interactions, was used by researchers to classify the repeated clinical alert as appropriate or inappropriate. Main Results: There were a total of 200 charts reviewed (male n = 61 (30.5%), female n = 139 (69.5%), mean age = 70 ± 11 years). Reasons for not contacting patients again include previous failure or adverse drug reaction (n = 62, 31.0%), patient did not meet call script requirements (n = 55, 27.5%), patient did not have diabetes or hypertension (n = 20, 10.0%), potential drug-disease interaction (n = 17, 8.5%), overlapping or previously addressed alerts (1.9%), or documentation was provided for “other” reasons (n = 43, 21.5%). The previous failure or adverse drug reaction rationale was appropriate in 32 of 62 repeated clinical alerts (52%; χ2= 10.15). The patient did not have diabetes or hypertension rationale was appropriate in 11 of 20 repeated clinical alerts (55%, χ2= 2.72). The potential drug-disease interaction rationale was appropriate in 3 of 17 repeated clinical alerts (8%, χ2= 9.89). The patient did not meet call script requirements rationale was appropriate in 31 of 55 repeated clinical alerts (56%, χ2= 6.91). The overlapping or previous alerts rationale was appropriate in 2 of 3 repeated clinical alerts (67%, χ2= 0.18). The “other” rationale were appropriate in 22 of 43 repeated clinical alerts (51%, χ2= 7.21) Overall, retrigger alerts were considered appropriate 50.5% of the time compared to the predicted value of 90% (χ2= 347 > critical value = 3.84 for p = 0.05 Conclusion: There are multiple reasons pharmacists do not recommend initiating ACEi therapy in patients with diabetes. Although the Medication Management Center (MMC) has rationale of these reasons documented after individual patient interactions, there are still several reasons why a retrigger alert would be appropriate despite that rationale. In addition, retrigger alerts were not considered appropriate as frequently as expected.

No description available.

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.