Les transporteurs vésiculaires du glutamate (VGLUTs) sont impliqués dans la recapture du glutamate du cytosol vers les vésicules présynaptiques. Depuis leurs caractérisations récentes en 2000, leurs implications dans plusieurs maladies neurodégénératives ont été démontrées. Ils jouent ainsi un rôle primordial dans la transmission nerveuse glutamatergique. Deux colorants naturels, le Rose Bengale et le Bleu Trypan, restent les meilleurs inhibiteurs connus à ce jour, avec respectivement des CI50 de 25 et 50 nM. Dans un premier temps, nous avons conçu et optimisé une série d’analogues basée sur le synthon Rose Bengale (inhibiteur non-compétitif). Ce travail a notamment permis de mettre en évidence l’effet des formes tautomères (quinone et lactone) sur l’inhibition des VGLUTs. Ainsi la forme quinonique, présente à pH physiologique, a été confirmée comme étant la seule capable de bloquer la recapture du glutamate. Dans un second temps, nous nous sommes intéressés à la famille du Bleu Trypan (inhibiteur compétitif) et nous avons déterminé la structure minimale active avec l’objectif de rendre ces molécules plus « drug-like ». En effet, l’intérêt de ce projet est de développer de petites structures aisément radiomarquables pour une utilisation dans un contexte physio-pathologique. / Vesicular glutamate transporters (VGLUTs) are involved in the recapture and storage of glutamate from cytol to secretory synaptic vesicules. Since their recent characterization in 2000, their implication in several neurodegenerative disorders have been demonstrated. They play a crucial role in glutamatergic neurotransmission. Natural dyes, such as Rose Bengal and Tryptan Blue are the best known inhibitors with IC50 values of 25 and 50 nM, respectively. Firstly, we designed and optimized a series of analogues based on the synthon Rose Bengal (non-competitive inhibitor). This work has especially enabled to highlight the effect of tautomeric forms (quinone and lactone) on the inhibition of VGLUTs. Thus, the quinone form, present at physiological pH, was confirmed as the only able to block the reuptake of glutamate. Secondly, we have been interested in the family of Trypan Blue (competitive inhibitor) and we determined the minimal active structure in order to render these molecules more "drug-like". Indeed, the interest of this project is to develop small structures easily radiomarquable to use in a physiopathological context.

The herein presented Ph.D. dissertation describes kinetic and structural characterization of human glutamate carboxypeptidases II and III (GCPII and GCPIII) using a complete panel of their natural substrates. These enzymes hydrolyze C-terminal glutamate from their substrates. They share 67 % sequence identity and also similar enzymatic activities. This thesis quantitatively compares human GCPII and GCPIII in terms of their ability to hydrolyze the substrates N-acetyl-L-aspartyl-L-glutamate (NAAG), folyl-poly-γ-L-glutamic acids (FolGlun) and β-citryl-L-glutamate (BCG). We demonstrated that GCPIII hydrolyzes its substrates in a metal- dependent manner, that BCG is a specific substrate of GCPIII, and that NAAG and FolGlun are specific substrates of GCPII. We also provide indirect biochemical evidence that GCPIII might feature a heterometallic active-site cluster. Additionally, we characterized the relevance of a surface exosite of GCPII, the arene-binding site (ABS), for the hydrolysis of FolGlun substrates using mutagenesis and enzyme kinetics and showed that polymorphic His475Tyr variant of GCPII hydrolyzes FolGlun substrates with the same kinetic parameters as the wild-type enzyme. Furthermore, this thesis focuses on structural aspects of the substrate specificities of GCPII and GCPIII: we present...

Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs) are omnipresent and persistent environmental pollutants. In particular, 29 congeners are of special concern, and these are usually referred to as dioxin-like compounds (DLCs). In the European Union, the risks associated with DLCs in food products are estimated by a weighted sum of the DLCs’ concentrations. These weights, also called toxic equivalency factors (TEFs), compare the DLCs’ potencies to the most toxic congener, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2378- TCDD). The toxicological effects of PCDD/Fs and PCBs are diverse, ranging from chloracne and immunological effects in humans to severe weight loss, thymic atrophy, hepatotoxicity, immunotoxicity, endocrine disruption, and carcinogenesis in rodents. Here, the molecular structures of DLCs were used as the basis to study the congeneric differences in in vitro data from both human and rodent cell responses related to the aryl hydrocarbon receptor (AhR). Based on molecular orbital densities and partial charges, we developed new ways to describe DLCs, which proved to be useful in quantitative structure-activity relationship modeling. This thesis also provides a new approach, the calculation of the consensus toxicity factor (CTF), to condense information from a battery of screening tests. The current TEFs used to estimate the risk of DLCs in food are primarily based on in vivo information from rat and mouse experiments. Our CTFs, based on human cell responses, show clear differences compared to the current TEFs. For instance, the CTF of 23478-PeCDF is as high as the CTF for 2378-TCDD, and the CTF of PCB 126 is 30 times lower than the corresponding TEF. Both of these DLCs are common congeners in fish in the Baltic Sea. Due to the severe effects of DLCs and their impact on environmental and human health, it is crucial to determine if other compounds have similar effects. To find such compounds, we developed a virtual screening protocol and applied it to a set of 6,445 industrial chemicals. This protocol included a presumed 3D representation of AhR and the structural and chemical properties of known AhR ligands. This screening resulted in a priority list of 28 chemicals that we identified as potential AhR ligands.

This thesis is engaged in the synthesis of polysubstituted pyrimidines with anti- inflammatory properties. Such molecules can inhibit production of prostaglandin E2 (PGE2). The aim of this study was to enhance water-solubility and anti-inflammatory efficacy of such derivatives via structural modifications of the lead scaffold. Among applied synthetic tools, the Suzuki-Miyaura cross-coupling was the prevalent reaction, however, many other synthetic procedures (Heck reaction, condensation, borylation, ozonolysis, nucleophilic substitution, etc.) were utilized as well. Overall, 43 final products were prepared. The anti-inflammatory efficacy (inhibition of PGE2 production) was successfully increased as the most potent compound achieved three orders of magnitude higher activity compared to the current lead structure WQE-134. Furthermore, no general influence of the length of the substituent in the C5 position of pyrimidine (C5pyr) on the anti-inflammatory efficacy of synthesized compounds was observed. Significant bioavailability obstacle in future development of the current lead WQE-134 is its poor solubility which was successfully enhanced by introduction of heteroatom bearing moieties to C5pyr. The most water-soluble compound achieved two orders of magnitude higher solubility than WQE-134 while...

Le carbonate de diméthyle (DMC) est utilisé comme intermédiaire en chimie des polymères. Actuellement, la production industrielle du DMC met en jeu un procédé polluant et dangereux. En revanche, la synthèse du DMC à partir de méthanol et de CO2 est un procédé « vert ». Cependant, la thermodynamique de la réaction est très défavorable, se traduisant par une conversion inférieure à 1%. L'objectif de cette thèse est de développer et d'étudier des catalyseurs très actifs qui, couplés à un réacteur membranaire, permettraient d'augmenter le rendement et l'activité. Ce manuscrit couvre plusieurs aspects : i) une étude de criblage de catalyseurs, ii) une étude de type structure-activité sur des séries de catalyseurs, basée sur des caractérisations structurales, texturales et de surface, iii) une étude mécanistique et iv) une étude cinétique. Un protocole de mesure d'activité adapté aux faibles conversions est développé. Le criblage catalytique met en évidence l'activité des solutions solides de cérine-zircone (CZ). Les CZ préparées par pyrolyse de flamme sont un ordre de grandeur plus actives que les CZ préparées par coprécipitation. De façon surprenante, il n'est pas possible a priori de distinguer des catalyseurs très actifs et peu actifs à partir de leurs caractéristiques structurales et texturales. Cependant, on observe que l'activité est corrélée à la densité et à la nature des espèces de surfaces méthoxides et carbonates. Le mécanisme réactionnel de l'état de l'art est affiné grâce à des mesures d'échanges isotopiques et de DRIFT. L'étude cinétique intègre des corrections thermodynamiques de l'équilibre de phases du binaire MeOH-CO2 qui constitue un liquide expansé / Dimethylcarbonate (DMC) is used in polymer synthesis as well as a fuel additive and solvent. The current industrial DMC production is a polluting and hazardous process. On the other hand, the direct carboxylation of methanol with CO2 is a green route to DMC. However, this reaction is highly limited by thermodynamics, limiting the conversion to less than 1%. The integration of a catalyst in a catalytic membrane reactor for water and DMC removal, would shift the equilibrium conversion thereby improving the DMC yield. The aim of this thesis is to develop and study highly active catalysts for DMC synthesis. This manuscript covers: (i) catalyst screening (ii) a structure-activity relationship study (iii) a mechanistic approach and (iv) a kinetic study. A protocol to measure the activity at low conversion has been developed. Catalyst screening evidenced solid solutions of ceria/zirconia (CZ) as the most active and selective. Flame sprayed pyrolysis ceria/zirconia are one order of magnitude more active than coprecipitated CZ. Interestingly, structural and textural features like crystalline and porous structure were similar regardless of the activity. Nevertheless, the activity could be correlated to the nature and the density of the methoxides and carbonates surface species. The mechanism was refined by isotopic exchange and DRIFT experiments. A kinetic study was performed in a batch reactor that integrated the physical equilibria of the gas-expanded reacting mixture

>Magister Scientiae - MSc / New therapeutic strategies are needed for a diverse array of poorly understood neurological impairments. These include neurodegenerative disorders such as Parkinson’s disease and Alzheimer’s disease, and the psychiatric disorders such as depression, anxiety and drug dependence. Popular neuropharmacotherapies have focused on dopamine (DA), serotonin (5HT), γ-aminobutric acid (GABA) and glutamate systems (Jupp & Lawrence, 2010). However recent research points to the sigma receptor (σR) as a possible neuromodulatory system. Due to its multi-receptor action, the σR can trigger several significant biological pathways. This indicates its ideal potential as a drug target to effectively minimise drug dosage and potential side effects. Currently there are a limited number of σR ligands available and few possess the selectivity to significantly show σR’s role in neurological processes. Polycyclic amines have shown notable sigma activity and provide an advantageous scaffold for drug design that can improve pharmacodynamic and pharmacokinetic properties (Banister et al., 2010; Geldenhuys et al., 2005). Aryl-heterocycle amine groups were also shown to improve σR activity (Piergentili et al., 2009).

A hemoglobina (Hb) é uma fonte reconhecida de peptídeos com funções biológicas diversas. O fragmento 33-61 da cadeia α da Hb, isolado do trato gastrointestinal do carrapato Boophilus microplus, foi o primeiro a ser descrito com ação antimicrobiana. O seu análogo sintético amidado, Hb33-61a, mostrou-se ativo contra bactérias Gram-positivas e fungos [Fogaça et al. (1999) J. Biol. Chem. 274, 25330-4]. O estudo de análogos do Hb33-61 nas formas amidada e com carboxila livre revelou que a amidação provoca aumento significativo da atividade frente a Candida albicans. Por apresentar propriedades biológicas e estruturais idênticas às do Hb33-61a, o Hb40-61a pareceu ser a sua porção mÌnima ativa [Sforça et al. (2005) Biochemistry 44, 6440- 51; Machado et al. (2007) Biopolymers 88, 413-26]. Para comprovar tal sugestão, no presente trabalho, sintetizamos, purificamos e caracterizamos novos an·logos do Hb33-61a, bem como os avaliamos quanto às suas atividades frente a C. albicans e Micrococcus luteus. Os resultados confirmaram a sugestão apenas para a ação antifúngica. O análogo Hb40-61a também se mostrou ativo frente a C. albicans resistente a fluconazol. A sua atividade antifúngica se mostrou fortemente dependente da força iônica do meio. A sua baixa atividade hemolítica foi confirmada mesmo em meio de baixa força iônica. O peptídeo Hb40-61a não apresentou sinergismo com o fluconazol frente a C. albicans. A cinética de morte celular mostrou que ele mata a levedura de forma rápida. Portanto, esta hemocidina sintética pode apresentar valor comercial se a via de administração for tópica ou se o seu uso envolver meios de baixa força iônica. Além disso, ela é um modelo valioso para o estudo do mecanismo de ação de peptídeos antimicrobianos com características estruturais similares e pode servir de base para o desenho de novos agentes antibiôticos. / It is well known that hemoglobin (Hb) is a source of biologically active peptides. The fragment 33-61 of bovine hemoglobin α-chain, isolated from the gut contents of the tick Boophilus microplus, was the first identified with antimicrobial activity . Its amidated analogue, Hb33-61a, showed to be active against Gram-positive bacteria and fungi strains [FogaÁa et al. (1999) J. Biol. Chem. 274, 25330-4]. The study of a series of carboxyl-free and amidated synthetic analogues of Hb33-61 revealed that C-terminus amidation enhances the activity against Candida albicans. Since Hb33-61a and Hb40-61a presented identical biological and structural properties, it seemed that Hb40-61a was Hb33-61a minimal active motif [SforÁa et al. (2005) Biochemistry 44, 6440- To test this suggestion, in the present study 51; Machado et al. (2007) Biopolymers 88, 413-26]. we synthesized, purified and characterized Hb40-61a analogues and assayed them against C. albicans and Micrococcus luteus. The results confirmed the suggestion only for the antifungal activity. When tested against fluconazole-resistant C. albicans, Hb40-61a was also active. Its antifungal activity showed to be dependent on the ionic strength of the medium. Its low hemolytic activity was confirmed even under low ionic strength conditions. Hb40-61a had no synergic effect with fluconazole on C. albicans. In vitro time-kill assays demonstrated that Hb40-61a kills the yeast rapidly. Therefore, this synthetic hemocidin may be of commercial interest for topical application or other uses involving low ionic strength medium. Moreover, it can serve as a template for the study of the mechanism of action of structurally related antimicrobial peptides or for the design of novel antibiotic drugs.

A reemergência de algumas bactérias Gram-positivas, em particular, do gênero Staphylococcus, como principal foco causador de infecções hospitalares, tem se intensificado nas últimas décadas, e, apesar da existência de potentes fármacos voltados para o tratamento de infecções causadas por este gênero de bactéria, as taxas de morbidade e mortalidade prevalecem com perfil crescente. Além disso, um grande problema associado a cepas de MRSA (Methicillin-Resistant Staphylococcus aureus) é o fenótipo de multi-resistência, característica que confere a este microrganismo resistência não apenas à meticilina como também a uma série de outros fármacos, exceto frente à vancomicina e à teicoplanina. Muito tem se feito, mas ainda são poucos os resultados efetivamente aplicáveis no tratamento de infecções com caráter de multi-resistência, justificando, desta forma, a necessidade de desenvolvimento de sucedâneos que sejam consideravelmente mais efetivos para a solução deste problema. Baseado nestes fatos, a proposta deste estudo envolveu o planejamento, síntese, identificação e estudos de QSAR (Quantitative Structure-Activity Relationships) em duas e três dimensões de derivados 5-nitro-2-tiofilidênicos com atividade antimicrobiana frente a cepas padrão e multi-resistente de Staphylococcus aureus. A escolha dos grupos substituintes foi realizada em duas etapas. Na primeira delas seguiu-se metodologia de substituição em anéis aromáticos proposta por Topliss para a otimização da bioatividade de compostos. Em uma segunda etapa, predominantemente quantitativa, foram selecionados mais alguns derivados baseando-se em faixa de hidrofobicidade ótima pré-determinada experimentalmente e na variação de efeito estérico dos grupos substituintes. Quatorze derivados 5-nitro-2-tiofilidênicos foram sintetizados, estruturalmente identificados e avaliados quanto à atividade antimicrobiana frente às cepas padrão (ATCC 25923) e multi-resistente (3SP/R33) de Staphylococcus aureus por determinação da concentração inibitória mínima empregando-se método de macrodiluição sucessiva em tubos. Salienta-se que a cepa 3SP/R33 se mostra resistente a dezenove antibióticos empregados na prática médica e apresenta suscetibilidade apenas à vancomicina. As concentrações inibitória e bactericida mínimas apresentadas pelos compostos sintetizados mostraram sofrer influência significativa da hidrofobicidade sobre as referidas atividades de acordo com os estudos de QSAR-2D e QSAR-3D, sendo os resultados obtidos para a cepa multi-resistente absolutamente compatíveis com os anteriormente determinados para a cepa padrão. Os estudos de QSAR-2D indicaram que a atividade antimicrobiana das 5nitro-2-tiofilideno benzidrazidas substituídas sofre influência significativa de duas propriedades físico-químicas que são a hidrofobicidade e a distribuição eletrônica. A relevância dos descritores estruturais σ e efe na determinação da atividade antimicrobiana, sinalizam que a distribuição eletrônica influencia fortemente o aumento da potência antimicrobiana dos compostos em estudo tanto pela influência dos efeitos indutivo e de ressonância na estrutura química do ligante, como também pelos campos moleculares gerados ao redor de grupos substituintes, sugerindo uma possível interação dos mesmos com uma área específica do sítio receptor. Nos estudos de QSAR-3D, foi evidenciado, em concordância com o estudo clássico anteriormente realizado, que a hidrofobicidade prevalece como propriedade de fundamental importância no estabelecimento da atividade antimicrobiana. Foi observada a importância da presença de regiões hidrofílicas pontuais nos compostos de forma a propiciar processos de solvatação e dessolvatação que são críticos na difusão através de membranas biológicas. Pode-se afirmar que a análise de QSAR, considerando os aspectos tridimensionais ligantes, ressaltou a necessidade de um balanço lipofílico-hidrofílico para um bom desempenho das 5-nitro-2-tiofilideno benzidrazidas ρ-substituídas como agentes antimicrobianos. A partir dos resultados obtidos evidenciou-se, neste estudo, o forte potencial de derivados 5-nitro-2-tiofilidênicos como possível alternativa para o desenvolvimento racional, em nível molecular, de fármacos voltados para o tratamento de infecções causadas por cepas multi-resistentes de Staphylococcus aureus. / In the last decade, there has been a reemergence of Gram-positive bacteria, in particular Staphylococcus, which isconsidered one of the. most causing of nosocomial infections. Although potent antistaphylococcal drugs are available, this infection continues presenting increasing morbidity and mortality rates. Besides, a serious problem associated with MRSA (Methicillin-Resistant Staphylococcus aureus) is the phenotype of multidrug resistance, which is, resistance not only to methicillin but also to many other drugs, except to vancomycin and teicoplanin. Many efforts have been made in a tentative to reduce this problem, nevertheless there is only a few number of alternatives to combat Staphylococcus aureus multidrug-resistant strains, justifying the necessity of development of more effective compounds to the treatment of these infections. Based in these facts, the purpose of this study was the design, synthesis, structural identification and 2D-QSAR and 3D-QSAR (Quantitative Structure-Activity Relationships) studies of 5-nitro-2-thiophylidene derivatives with antimicrobial activity against multidrug-resistant strains of Staphylococcus aureus. The choice of substituent groups was made in two stages. The first stage comprises on application of Topliss operational scheme for aromatic substitution. In a second quantitative stage, more derivatives were selected according by hydrophobicity range previously determined. Other standard considered at the selection of substituent groups was the variation of steric effect. Fourteen 5-nitro-2-thiophylidene derivatives were synthesized, structural identified and tested against standard (A TCC 25923) and multidrug-resistant (3SP/R33) strains of Staphylococcus aureus. The Minimal Inhibitory Concentration, MIC, was determined using the serial dilution tests in two sequential stages. The 3SP/R33 strain is resistant to nineteen antimicrobial agents in use, except to vancomycin. The minimal inhibitory and bactericidal concentrations of synthesized compounds showed, according by 2D-QSAR and 3D-QSAR studies, a significant influence of hydrophobic properties on antimicrobial activity determination and the results obtained for multidrug-resistant strain were consistent with those determined for A TCC 25923 strain. 2D-QSAR studies showed that antimicrobial activity are mainly influenced by two physico-chemical properties: hydrophobicity and electronic distribution. The relevance of σ e ephe parameters on antimicrobial activity determination, denotes the contribution of inductive and resonance effects for the polar performed by the substituent groups, probably suggesting an interaction between them and specific receptor site. 3D-QSAR studies showed that hydrophobicity is a essential property to antimicrobial activity determination, sustained the same conclusions previously obtained by Hansch Analysis. It was observed a great concern of small hydrophilic regions distributed on derivatives in order to promote solvation and desolvation process, that have critical importance on diffusion process through the biological membranes. QSAR studies considering three-dimensional properties of ligands indicated the necessity of accurate hydrophilic-hydrophobic balance on nitrothiophene derivatives for their good performance as antimicrobial agents. The results obtained in this preliminary study have shown the potential of synthesized compounds as alternatives to the treatment of infections caused by multidrug-resistant strains of Staphylococcus aureus.

Ce travail de thèse a porté sur l’étude structurale de peptides urotensinergiques humains par DC, RMN etmodélisation moléculaire. L’hUII (11 aa) et son analogue l’URP (8 aa) sont considérés comme les peptides vasoactifs les plus puissants connus à ce jour et sont impliqués dans divers systèmes biologiques, notamment le système cardiovasculaire et la prolifération des cellules tumorales. Ces deux peptides sont des ligands endogènes d'un RCPG, l’UT. Ils peuvent exercer des actions physiologiques communes mais aussi divergentes. Afin d’apporter des éléments permettant une meilleure compréhension de leurs activités biologiques, nous avons, dans un premier temps, déterminé la structure 3D de trois agonistes (hUII4-11, URP,P5U) et d’un antagoniste (urantide) dans un milieu micellaire mimant les membranes des cellules eucaryotes, le DPC. Dans les quatre peptides, nous avons observé la présence de deux conformations majoritaires du pont disulfure, RHStaple et LHHook, qui sont connues pour être essentielles à l’activité biologique. Nous avons mis en évidence une différence de nature de coude entre les agonistes (coude β de type I) et l’antagoniste (coude β de type II’). Nos analyses ont également permis de montrer l’existence de variations d’orientation des chaînes latérales des résidus F6, Y9 et plus spécialement celle de W7 entre les agonistes etl’antagoniste. Le groupe indole du D-W7 présente ainsi une rotation de 180°. Dans un deuxième temps, nous avons mis en évidence un impact de la concentration sur la conformation de l’hUII qui n’est pas observé pour l’URP. Ce phénomène d’auto-association pourrait avoir une influence sur l’interaction avec le récepteur et être à l’origine des divergences d’activités biologiques entre l’hUII et l’URP. / This work aims to characterize the structure of human urotensinergic peptides by CD, NMR and molecular modelling. hUII (11 aa) and its analogue URP (8 aa) are considered as the most potent vasoactive peptides known so far and are involved in various biological systems, including the cardiovascular system and tumor cell proliferation. These two peptides are endogenous ligands of a GPCR, UT, and exert common but also divergent physiological actions. In order to gain a better understanding of their biological activities, we determined the structures of three agonists (hUII4-11, URP, P5U) and one antagonist (urantide), in DPC micelles, a cellular eukaryotic mimetic membrane. For all peptides, we observed the presence of two major forms of the disulfide bridge, RHStaple and LHHook, which are known to be essential for biological activity. We showed a difference in the turn nature between agonists (type I β turn) and the antagonist (type II’ β turn). Our analyses also revealed that, in agonists and antagonist, the side chain orientations of residues F6, Y9 and more specifically W7 were different. Indeed, the indole group of D-W7 exhibited a 180° rotation. Secondly, we showed that, contrary to URP, the conformation of hUII was dependent on concentration. This selfassembly phenomenon may impact the interaction with the receptor and be responsible for the differential biological activities of hUII and URP.

N-methyl-D-aspartate (NMDA) receptors are glutamate-gated calcium permeable ion channels that play a key role in excitatory synaptic transmission and plasticity, and their dysfunction underlies several neuropsychiatric disorders. The overactivation of NMDA receptors by tonically increased ambient glutamate can lead to excitotoxicity, associated with various acute and chronic neurological disorders, such as ischemia, Alzheimer and Parkinson's disease, epilepsy or depression. On the opposite, NMDA receptor hypofunction is thought to be implicated in autism, schizophrenia, or intellectual disability. Recent DNA screening for neurological and psychiatric patients revealed numerous mutations in genes encoding for NMDA receptor subunits. The activity of NMDA receptors is influenced by a wide variety of allosteric modulators, including neurosteroids that could both inhibit and potentiate the activity of NMDA receptors, which makes them promising therapeutic targets. In this thesis, we describe new classes of neurosteroid analogues which possess structural modifications at carbons C3 and C17 of the steroidal core, and analogues without D-ring region (perhydrophenanthrenes). We evaluated the structure-activity relationship (SAR) for their modulatory effect on recombinant GluN1/GluN2B receptors. Our results...