Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (M.S.)--University of Michigan, 1989.

Thesis (M.S.)--University of Michigan, 1989.

Tissue transglutaminase (TG2) is a calcium-dependent enzyme that natively catalyses the formation of isopeptidic bonds between protein- or peptide-bound glutamine and lysine residues. Physiologically, it is ubiquitously expressed in tissues, with roles in cellular differentiation, extracellular matrix stabilisation, and apoptosis, among others. However, its unregulated activity has been associated with various pathologies including fibrosis, cancer and celiac disease. Since most pathologies are associated with an increased transamidation activity, efforts have been directed towards the development of TG2 inhibitors. In this context, the work described in this thesis is centred on reversible inhibitors, building on recent work done within the Keillor group in two directions, namely localisation and potency. In a localisation-driven approach, we developed a photolabile derivative of a known reversible inhibitor, in order to form a covalent bond with the enzyme and determine the inhibitor’s binding site. In tandem, we optimised a protocol for the expression of TG2 incorporating ArgΔ10 and LysΔ8, amino acids that are 13C- and 15N-labelled to provide a mass shift of 10 and 8 Da, respectively, compared to the corresponding unlabelled amino acids. This “heavy” TG2 was developed as a tool for reference in the analysis of the tryptic digest of labelled protein. In a potency-driven approach, based on the observation that previous trans cinnamoyl inhibitor scaffolds were susceptible to nucleophilic attack by glutathione, we developed a bis(triazole) scaffold with reduced electrophilicity. The preparation of a small library of compounds showed that this scaffold demonstrates a preference for electron-withdrawing substituents, such as nitro groups. Continuing in a potency-driven approach, and inspired by work done in the identification of glutathione-resistant scaffolds, we studied a new alkynyl scaffold. While still susceptible to glutathione addition, these compounds showed a marked improvement in potency, with the lead compound having an IC50 of 930 nM and being established as a competitive inhibitor with a Ki of 420 nM, our most potent reversible inhibitor to date. Furthermore, this scaffold also produced an inhibitor lacking nitro groups (to limit eventual cellular toxicity), but maintaining good potency, with an IC50 value of 3.03 μM.

Nous nous sommes inspirés des solutions élégantes que la Nature propose, concernant le contrôle et l’optimisation de réactions spécifiques, pour présenter une tentative d’imitation des interfaces biocatalytiques au niveau de nanotubes. Des brosses de nanotubes auto-assemblés à l’aide d’un composé enzymatique, la bêta-lactamase, ont été préparées via des techniques de nanofabrication comme le «layer-by-layer» et le «hard templating».En premier lieu, les effets de confinement géométriques et ses conséquences ont été étudiés et comparés pour des assemblages de films de chitosan/bêta-lactamase sur des surfaces planes et au sein de membranes nanoporeuses.Ensuite, des nanotubes de polyélectrolytes de dimensions contrôlées ont été préparés dans des membranes nanoporeuses puis ancrés sur une surface par couplage chimique pour obtenir des brosses de nanotubes. Des études cinétiques révèlent la présence d’enzymes actives dans ces brosses et une amélioration de la préservation de l’activité quand la bêta-lactamase a été déposée dans les couches intérieures des nanotubes.Enfin, une variété de couches thermo-sensibles avec différentes architectures a été testée pour contrôler la diffusion du substrat sur les films multicouches de bêta-lactamase. L’intégration d’éléments thermo-sensibles stables a été prouvée. Mais des expériences complémentaires avec des mécanismes plus complexes tels que le couplage des réponses thermiques et mécaniques sont nécessaires pour contrôler la biocatalyse impliquant des couches supplémentaires.En résumé, cette étude présente des éléments ouvrant la voie à l’intégration de techniques pour la fabrication de nanostructures complexes pour la biocatalyse. / Inspired by the elegant solutions that Nature has provided to control and promote specific site-reactions, my work presents an attempt to mimic filamentous biocatalytic interfaces. Brushes of self-assembled nanotubes with an enzymatic component (beta-lactamase) were prepared taking advantage of preexisting nanofabrication techniques, such as layer-by-layer and hard-templating.First, the effects of geometrical confinement and its consequences were investigated by comparison of (chitosan/beta-lactamase) multilayer film assembly on flat surfaces and in nanoporous membranes. In a second stage, polyelectrolyte nanotubes with controlled dimensions were prepared in nanoporous membranes and further anchored on a surface by chemical crosslinking to obtain brushes of nanotubes. The kinetic studies revealed the presence of active enzyme in the brushes and enhanced activity preservation when beta-lactamase was deposited as the inner layers of the nanotubes.As a final step, a variety of thermo-responsive coatings with different architectures were tested to control substrate diffusion on top of beta-lactamase-based multilayer films. The integration of stable thermo-responsive elements was proven, although further experiments are required to control biocatalysis with additional layers and using more complex mechanisms, such as coupled thermal and mechanical responses. Knowing that there are more challenges to face before reaching optimum nanotube brushes and apply them for controlled biocatalysis, this study contributes with some elements that may pave the way towards the integration of different techniques for the fabrication of complex biocatalytic nanostructures.

The nucleus may be the critical site for the activation of chemical carcinogens, and subsequently the initiation of neoplasia. However, isolated nuclei may be contaminated with endoplasmic reticulum, the major site of the drug metabolizing enzymes. One of the objectives of the present study was to determine whether the enzymes in isolated rat hepatic nuclei were of nuclear origin and, if so, to compare these enzymes with those in the microsomal fraction. The selective manipulation of nuclear enzymes would be a useful tool in determining their role in cellular toxicity. Recentrifugation experiments, with aryl hydrocarbon hydroxylase (AHH) activity as a marker, showed that isolated nuclei were not contaminated with endoplasmic reticulum in the form of microsomes formed upon homogenization. However, small "tags" of endoplasmic reticulum, continuous with the nuclear membrane, and indiscernable in electron micrographs, could remain following centrifugation and account for all of the measurable enzyme activity in the isolated nuclei. It was reasoned that if endoplasmic reticulum accounted for all of the activity, then the ratio of nuclear to microsomal activity for all enzymes determined should be the same. The ratios of epoxide hydrolase and AHH were found to differ in the two fractions. The simplest interpretation of these data was that drug metabolizing enzymes existed in the nuclei. However, the distribution of drug metabolizing enzymes throughout the endoplasmic reticulum is known to be heterogeneous and these "tags" could differ from the total endoplasmic reticulum (microsomes) in their enzyme make-up. Whether these enzymes are in the nuclear membrane, nucleoplasm, or as "tags" of endoplasmic reticulum, they represent activities in close proximity to potential target sites in the nucleus. The inhibition, induction, and activation characteristics of nuclear and microsomal enzymes were studied with the goal of selective manipulation of nuclear enzymes. The enzymes in the nuclear and microsomal fractions were found to differ' only in quantitative inducibility, and were identical in all other respects. Therefore, the selective manipulation of nuclear enzymes was not achieved. The induction of hepatic drug metabolizing enzymes is a measure of altered genetic expression in the liver. Inducers of drug metabolizing enzymes have also been shown to promote neoplasia in the liver. Therefore, studying the induction of such enzymes may lead to a further understanding of the mechanism of tumour promotion. Phenobarbital, 3-methylcholanthrene and pregnenolone-16α-carbonitrile produce three distinct induction responses. In the present study, spironolactone and trans-stiIbene oxide were shown to produce distinct induction responses, also. Spironolactone was shown to be a different inducer based on the protein band patterns observed following SDS-polyacrylamide gel electrophoresis of liver microsomes. trans-Stilbene oxide was found to produce a significantly different maximal level of AHH activity. The observation of five distinct induction responses suggests at least five recognition sites (receptors) mediating the pleiotropic actions of exogenous compounds in the liver. / Pharmaceutical Sciences, Faculty of / Graduate

Le développement du parasite Plasmodium falciparum, responsable du paludisme, nécessite la synthèse de phospholipides et plus particulièrement de phosphatidylcholine (PC) et phosphaditylethanolamine (PE) qui représentent environ 85% de la totalité des phospholidipes du parasite. Leur synthèse s'effectue principalement par les voies métaboliques de novo, voies de Kennedy, en trois étapes enzymatiques. Les enzymes CTP: phosphoethanolamine cytidylyltransferase (ECT) et CTP: phosphocholine cytidylyltransferase (CCT) catalysent les étapes limitantes des deux voies de biosynthèse de la PE et de la PC, respectivement. Ces deux enzymes sont essentielles à la survie du parasite murin, P. berghei et représentent ainsi des cibles thérapeutiques potentielles. La PfCCT est constituée de deux domaines cytidylyltranférases (CT) répétés alors que l'enzyme homologue chez l'homme est composée d'un seul domaine. En revanche, pour la ECT, la présence de deux domaines CT est retrouvée chez toutes les espèces mais les analyses de séquences et de structures ont montré que des résidus importants du site catalytique liant le substrat n'étaient pas conservés dans le domaine CT C-terminal de la PfECT. Ce travail a eu pour but de déterminer les propriétés enzymatiques et les caractéristiques cellulaires de la PfECT et de la PfCCT. Les paramètres cinétiques de ces enzymes ont été quantifiés in vitro à l'aide protéines recombinantes ainsi que sur les enzymes endogènes à l'aide d'extraits parasitaires. Grâce à l'utilisation de protéines recombinantes ponctuellement mutées, nous avons montré que seul le domaine CT N-terminal de la PfECT est catalytiquement actif. Chez P. falciparum, la PfECT et la PfCCT sont exprimées tout au long du cycle intra-érythrocytaire du parasite. La PfECT est présente dans la fraction soluble du parasite alors que la PfCCT apparait aussi bien dans la fraction soluble qu'insoluble. Des expériences d'immunofluorescence ont montré que la PfECT est cytosolique. L'ensemble des résultats présentés apportent un éclairage important sur les fonctions et les propriétés de ces deux cibles potentielles et constituent les premières étapes indispensables à l'élaboration d'une approche thérapeutique. / Phospholipids are essential for the growth and development of Plasmodium falciparum malaria parasite. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are its major structural phospholipids. This study focused on CTP: phosphoethanolamine cytidylyltransferase (ECT) and CTP: phosphocholine cytidylyltransferase (CCT) that catalyzes the rate-limiting steps of the de novo Kennedy pathways for PE and PC biosynthesis respectively. Both ECT and CCT are essential in the rodent malaria parasite P. berghei and constitute potential chemotherapeutic targets to fight against malaria. PfCCT consists of two very similar cytidylyltransferase (CT) domains whereas the human enzyme consists of only one CT domain. The presence of two CT domains in ECT seems to be widespread in all the organisms. Sequence and structural analysis showed that the C-terminal CT domain of ECT lacks key residues in the substrate binding motif. This study aimed at unravelling the enzymatic properties and cellular characteristics of PfECT and PfCCT enzymes. In addition, these studies addressed the key question if C-terminal CT domain of PfECT is catalytically active. Kinetic parameters of the enzymes were evaluated in vitro on native proteins as well as on recombinant proteins, the latter being produced in bacterial system. Cellular characterisation studies using polyclonal antisera showed that PfECT and PfCCT are expressed throughout the intra-erythrocytic life cycle of the parasite. PfECT is found mainly in soluble form in the parasite while PfCCT is present in soluble as well as insoluble forms in the parasite. Furthermore, immunofluorescence studies for PfECT revealed that it is mainly cytosolic. To assess the contribution of each CT domain to overall PfECT enzyme activity, recombinant PfECT mutants were generated by site-directed mutagenesis. Kinetic studies on these mutants indicated that the N-terminal CT domain was the only active domain of PfECT. Collectively, these results bring new insights into the kinetic and cellular properties of the enzymes and will pave the way in developing a future pharmacological approach.

Les leishmanioses sont des maladies tropicales négligées provoquées par un protozoaire parasite du genre Leishmania, et transmises par un insecte vecteur, le phlébotome. Les leishmanioses menacent 310 millions de personnes dans 98 pays à travers le monde. Les traitements antileishmaniens actuels sont limités et présentent des problèmes majeurs de toxicité et d'émergence de chimiorésistance. Dans ce contexte, il est nécessaire de développer de nouveaux agents antileishmaniens spécifiquement dirigés contre une cible thérapeutique chez le parasite. La GDP-Mannose Pyrophosphorylase (GDP-MP) est une cible thérapeutique essentielle à la survie du parasite à la fois in vitro et in vivo. Plusieurs différences ont été identifiées dans le site actif de GDP-MPs leishmaniennes par rapport à l'enzyme humaine, montrant ainsi l'intérêt de cette cible thérapeutique dans le développement de nouveaux traitements contre la leishmaniose. La GDP-MP catalyse la synthèse du GDP-mannose, la forme activée du mannose, brique moléculaire importante dans les processus de glycosylation et la synthèse de glycoconjugués essentiels à la reconnaissance hôte-parasite. Ce travail de thèse a consisté à produire et purifier les GDP-MPs de 3 espèces de parasites (Leishmania infantum, Leishmania donovani et Leishmania mexicana) ainsi que l'homologue humaine dans le but de comparer leurs propriétés enzymatiques. A partir d'inhibiteurs potentiels conçus et synthétisés sur la base de modèles moléculaires de GDP-MPs leishmaniennes et humaine, 100 composés ont été évalués sur les enzymes purifiées et sur les parasites in vitro. Cette analyse nous a permis de sélectionner des composés spécifiquement dirigés contre la cible du parasite et présentant une activité antileishmanienne. Nous avons également initié une étude de l'expression et de la localisation de la GDP-MP après traitement par les composés les plus intéressants. Ces composés pourront par la suite être utilisés comme outils pharmacologiques pour le développement de nouveaux agents antileishmaniens spécifiques. / Leishmaniases are Neglected Tropical Disease (NTD)caused by a protozoan parasite of the genus Leishmania and transmitted by an insect vector, the phlebotomine sandfly. Leishmaniases threaten 310 millions people in 98 countries around the world. Current antileishmanial treatments are limited and present major issues of toxicity and drug resistance emergence. In this context, it is necessary to develop new specific antileishmanial drugs specifically directed against a therapeutic target in the parasite.The GDP-Mannose Pyrophosphorylase (GDP-MP) is a therapeutic target which has been described to be essential for parasite survival both in vitro and in vivo. Several differences have been identified in the active site of leishmanial GDP-MPs compared to the human counterpart, showing the prominence of this therapeutic target in the development of new treatments against leishmaniasis. The GDP-MP catalyzes the synthesis of GDP-Mannose,the activated form of mannose, an important molecular constituent of the glycosylation processes involved in the biosynthesis of glycoconjugates which are essential for host-parasite recognition. My thesis work consisted in the production and purification of GDP-MPs from 3 Leishmania species (Leishmania donovani,Leishmania infantum and Leishmania mexicana)and from humanin order to compare their enzymatic properties. From potential inhibitors designed and synthesized on the basis of leishmanial and human GDP-MP molecular models, 100 compounds were evaluated on purified enzymes and on parasites in vitro. These analyses allowed us to select some compounds which are specifically directed against the parasite target and presenting antileishmanial activities. We have also initiated a study of expression and localization of GDP-MP after treatment with the most potent compounds. These compounds will be used as pharmacological tools for the development of new specific antileishmanial drugs.

The enzyme soluble epoxide hydrolase (sEH) plays a major role in the pathogenesis and pathophysiology of neurodegenerative diseases like depression by catalyzing the hydrolysis of epoxyeicosatrienoic acids (EETs) into dihydroxyicosatrienoicacids (DHETs), its less biologically active form, influencing the anti-inflammatory system and promoting inflammation. Therefore, inhibiting sEH leads to increased levels of EETs, reducing inflammation, especially in the brain and can help mitigate neurodegenerative diseases. This study investigated sEH inhibition by a phenolic carotenoid compound, astaxanthin and its inhibitory mechanism of action. Enzyme inhibitory activity and kinetics demonstrated that astaxanthin had a half-maximal inhibitory concentration (IC50) of 26 ± 0.92 μM and is a mixed-non-competitive inhibitor of sEH. In silico ADME/tox analysis showed that astaxanthin is bioavailable, biostable, and non-toxic when taken orally. Molecular docking study demonstrated that astaxanthin binds to an allosteric site of sEH and formed a contact and clashing-only interaction with the ASP333 residue of the hydrolase pocket of sEH. In this study, we highlight the potential therapeutic application of astaxanthin as a natural sEH inhibitor in the treatment of inflammation-related diseases, particularly neurodegenerative diseases.

Global climate change is identified as a major threat to the survival of natural ecosystems. The variations in global climate have gained the attention of researchers worldwide, as these changes negatively affect agriculture by reducing crop productivity and food security.  Projects related to abiotic stress tolerance are significant because they address important challenges facing agriculture and food security, contribute to more sustainable agricultural practices, and advance our understanding of fundamental plant biology. Some plants have defense mechanisms that are activated upon receiving stress stimuli to increase systemic tolerance to abiotic stresses such as heat, light, and cold. Salicylic acid-binding protein 2 (SABP2) from tobacco exhibits a high affinity for salicylic acid (SA) and is an important component in the SA-signaling pathway. SABP2 interacts with other cellular proteins to initiate downstream signaling and activate responses leading to resistance. Several SABP2-interacting proteins (SIP), including SIP-428, have been identified. The main goal of this proposed research is to determine the role of SIP-428 in mediating environmental stresses. SIP-428 is a SIR2-type non-histone deacetylase enzyme. De/acetylation is a common post-translational modification of proteins in eukaryotes. Since SIP-428 is a SABP2-interacting protein, it is involved in plant immune signaling. To determine the role of SIP-428 in plant physiology, it was biochemically characterized, and transgenic tobacco plants silenced in SIP-428 expression were previously generated and analyzed. Transgenic tobacco plants overexpressing SIP-428 were also generated. These lines expressed SIP428 at higher levels upon treatment with estradiol. Transgenic tobacco that overexpresses SIP-428 has been used in this study. To test the role of SIP428 in abiotic stress, the transgenic plants will be treated with abiotic stress-inducing chemicals, e.g. NaCl (salinity stress), mannitol (osmotic stress), and PEG6000 (drought stress). The treated seedlings will be allowed to grow for a specific time (1-2 weeks). The expression of SIP-428 will be monitored by western blotting (using anti-myc antibodies). The effects of SIP-428 expression on abiotic stress tolerance will be investigated biochemically by examining the activities of antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and ascorbate peroxidase (APX). Additionally, gene expression analysis will also be conducted to determine the expression of antioxidant genes.