Evolution of enzymes with novel functional properties has gained much attention in recent years. Naturally evolved enzymes are adapted to work in living cells under physiological conditions, circumstances that are not always available for industrial processes calling for novel and better catalysts. Furthermore, altering enzyme function also affords insight into how enzymes work and how natural evolution operates. Previous investigations have explored catalytic properties in the directed evolution of mutant libraries with high sequence variation. Before this study was initiated, functional analysis of mutant libraries was, to a large extent, restricted to uni- or bivariate methods. Consequently, there was a need to apply multivariate data analysis (MVA) techniques in this context. Directed evolution was approached by DNA shuffling of glutathione transferases (GSTs) in this thesis. GSTs are multifarious enzymes that have detoxication of both exo- and endogenous compounds as their primary function. They catalyze the nucleophilic attack by the tripeptide glutathione on many different electrophilic substrates. Several multivariate analysis tools, e.g. principal component (PC), hierarchical cluster, and K-means cluster analyses, were applied to large mutant libraries assayed with a battery of GST substrates. By this approach, evolvable units (quasi-species) fit for further evolution were identified. It was clear that different substrates undergoing different kinds of chemical transformation can group together in a multi-dimensional substrate-activity space, thus being responsible for a certain quasi-species cluster. Furthermore, the importance of the chemical environment, or substrate matrix, in enzyme evolution was recognized. Diverging substrate selectivity profiles among homologous enzymes acting on substrates performing the same kind of chemistry were identified by MVA. Important structure-function activity relationships with the prodrug azathioprine were elucidated by segment analysis of a shuffled GST mutant library. Together, these results illustrate important methods applied to molecular enzyme evolution.

A screening method was developed for identification of catalytically active enzymes in combinatorial cDNA libraries of mutated glutathione transferase (GST) derivatives expressed in E. coli. The method is based on spraying monochlorobimane (MCB) directly over bacterial colonies growing on agar. The substrate MCB become fluorescent under UV light, when the bacterial colony contains active GSTs catalyzing the conjugation with endogenous glutathione. Eleven out of twelve GSTs investigated where active with MCB. This method can be used to screen libraries generated from most cytosolic GSTs in the search for proteins with altered functions and structures. Azathioprine (Aza), a thiopurine that has been used clinically for 40 years was investigated with 14 GSTs. Three enzymes showed prominent catalytic activities with Aza and all of them are highly expressed in the liver. We estimated the contribution of the three enzymes GSTs A1-1, A2-2 and M1-1 bioactivation of Aza in the liver and concluded that it was about 2 orders of magnitude more effective than the uncatalyzed reaction. GST bioactivation of Aza could clarify aspects of idiosyncratic reactions observed in some individuals. Two other thiopurine prodrugs, cis-acetylvinylthiopurine (cAVTP) and trans-acetylvinylthioguanine (tAVTG), were investigated with the same 14 GSTs. The results displayed diverse catalytic activities. A mechanism of consecutive reactions was proposed. The studies contribute to knowledge under what conditions the drug should optimally be administered. A study of the same prodrugs with several mutants from the Mu class characterized by a point mutation of a hypervarible residue. We conclude that the effects of the mutations were qualitatively parallel for cAVTP and tAVTG, but they vary significantly in magnitude; steric hindrance may interfere with transition-state stabilization. From the evolutionary perspective the data show that a point mutation can alternatively enhance or attenuate the activity with a particular substrate and illustrate the functional plasticity of GSTs.

Thesis (Ph. D.)--Ohio State University, 2007.

A doença de Chagas, parasitose endêmica causada pelo Trypanosoma cruzi, se apresenta como grande causa de morbimortalidade, afetando cerca de 18 milhões de pessoas no continente americano e causando 21.000 mortes, a cada ano. Atualmente, estima-se que 100 milhões de pessoas estejam sob risco de contaminação nos 18 países da área endêmica da doença. A quimioterapia contra a tripanossomíase americana é constituída por apenas dois fármacos, nifurtimox e benznidazol, que não apresentam ação adequada na fase crônica da doença. Por estas razões, é premente a necessidade de novas e mais eficazes alternativas terapêuticas. A cruzaína, mais abundante cisteíno-protease do parasita, é enzima essencial em todos os estágios de modificação celular do parasita e está, também, presente no processo de invasão e modificação do sistema imune do hospedeiro. Além disso, apresenta diferenças em relação a enzimas dessa categoria no hospedeiro. Trata-se, pois, de excelente alvo bioquímico para a pesquisa de novos agentes contra o T. cruzi. Face ao exposto e tendo-se em vista a especificidade da cruzaína, considera-se de grande interesse a compreensão do mecanismo de interação de compostos com essa enzima com o intuito de planejar derivados com atividade antichagásica potencial. O presente trabalho teve o objetivo de elucidar a afinidade de diferentes classes de compostos pela enzima, na busca racional de novos tripanomicidas. Assim, pró-fármacos peptídicos recíprocos, derivados de primaquina(PQ) e de nitrofural(NF), anteriormente sintetizados, e pró-fármacos peptídicos duplos do hidroximetilnitrofural (NFOH) planejados, e cuja síntese foi estudada, foram submetidos a estudos de modelagem molecular e de docking. Os peptídios foram escolhidos com base na cisão específica pelacruzaína. Avaliou-se a interação com a enzima e elucidar o mecanismo de liberação dos fármacos e composto ativo a partir desses derivados. Os estudos indicaram que o melhor transportador a ser utilizado no planejamento de novos pró-fármacos é o dipeptídio LysArg, corroborando o que havia sido observado nos ensaios em cultura de células infectadas . com o T. cruzi. Ante a possibilidade de um segundo mecanismo de ação pela interação entre a Cys25 da cruzaína e o grupo semicarbazona, presente no NF e no NFOH, planejaram-se e sintetizaram-se análogos destes compostos, utilizando bioisosterismo clássico entre enxofre e oxigênio. Estudos de modelagem molecular e docking indicaram a participação também deste mecanismo de ação na atividade dos referidos derivados nitro-heterocíclicos. Todos os bioisósteros apresentaram ação inibitória na cruzaína IC50 entre 2,71 µM e 22,83 µM - evidenciando, também, este mecanismo de ação. Com o objetivo de se estudar a influência do grupamento (tio)semicarbazona presente nos derivados bioisostéricos sintetizados, realizaram-se estudos de QSAR 20 e 3D em série de compostos descritos na literatura, obtendo-se modelos robustos e com grau elevado de predição, comprovando a ação na cruzaína. Tais grupos podem ser utilizados no planejamento de novos tripanomicidas. Complementando a busca racional de novos antichagásicos potenciais, efetuou-se triagem virtual de novos ligantes utilizando o planejamento racional com base na estrutura do receptor (SBDD) a partir de modelo farmacofórico específico obtido para a cruzaína. Esse estudo proporcionou a sugestão de vinte moléculas a partir do banco de dados CHEMDIV, com possível ação inibitória da enzima. Com base nos resultados obtidos, conclui-se que os estudos realizados com pró-fármacos e análogos por meio de processos racionais de planejamento forneceram dados importantes para a pesquisa de candidatos a novos fármacos antichagásicos. / Chagas \' disease, a parasitosis caused by Trypanosoma cruzi, is an endemic disease that affects most part of Latin America. About 18 million people are infected by the parasite and around 21 thousand deaths are related to Chagas\' disease each year. Nowadays, 100 millions of people are estimated to be under the risk of infection in the 18 countries of the endemic areas. The therapeutic armamentarium available against Chagas\' disease is comprehended by only two drugs, nifurtmox and benznidazol, which are not effective in the chronic phase of the disease. Cruza in, the most abundant cysteine protease of the parasite, is essential in all stages of the cellular development of the parasite and responsible for invasion and modification of the immunologic system of the human host. Besides, it has differences relatively to those enzymes in the humans. So, it is an excellent biochemical target for searching new agents against T. cruzi. This said and in the view of cruzain specificity, understanding the mechanism of interaction of compounds with this enzyme is considered very interesting in order to design derivatives with potential anti-Chagas\'disease. The present work had the objective of elucidating the affinity of different classes \'of compounds to the enzyme, in the rational search for new trypanomicides. So, mutual peptide prodrugs, derived from primaquine (PQ) and nitrofurazone (NF), previously synthesized, and designed double peptide prodrugs of hydroxymethylnitrofurazone (NFOH), which synthesis has been studied, were submitted to molecular modeling and docking studies. The peptides were chosen based on specific cleavage by cruzain. The interaction with the enzyme and the drug as well as the active compound release mechanism from those derivatives were evaluated. The studies have indicated the dipeptide LysArg as the best carrier to be used in the design of new prodrugs, corroborating what had been earlier observed in the tests of T. cruzi infected cell culture. Based on the possibility of a second mechanism of action through the interaction between cruzain Cys 25 and the semicarbazone group, found in NF and NFOH, analogs of this c/ass of compounds were designed, and synthesized, using classic bioisosterism between sultur and oxygen. Molecular modeling and docking studies have indicated also the participation of this mechanism in the activity of the nitro-heterocyclic compounds referred. All bioisosters showed to inhibit cruzain IC50 between 2.71 µM and 22.83 µM -- also evidencing this mechanism of action. With the purpose of studying the influence of (thio)semicarbazone group present in the synthesized bioisoster derivatives, 20 and 3D QSAR have been developed for a series of compounds reported in the literature, leading to robust and high- preditive leveI models, confirming their action in cruzaine. Those groups might be used in the design of new trypanomicides. Complementing the rational search for new antichagasic compounds, a virtual screening of new ligands, using the structure-based drug design (SBDD) was developed based on a specific pharmacophore model obtained for cruzain. This study have provided the suggestion of twenty compounds from the CHEMDIV data bank with possible inhibitory activity in cruzain. Based on the results obtained, the conclusion is that the studies herein developed with prodrugs and analogs through rational drug design lead to important data towards the research of new candidates as new antichagasic drugs.

Doenças cardiovasculares podem ocasionar manifestações clínicas graves como infarto agudo do miocárdio e acidentes vasculares trombóticos, constituindo a principal causa de morte no mundo, fato esse que desperta grande interesse da indústria farmacêutica. As causas normalmente estão relacionadas à elevação dos níveis de colesterol e à agregação plaquetária, que acarretam eventos vaso-oclusivos. Entre as alternativas terapêuticas para o controle e prevenção das doenças cardiovasculares podem-se destacar os inibidores da 3-hidroxi-3-metilglutaril coenzima-A redutase (HMG-CoA redutase), popularmente conhecidos como estatinas. A rosuvastatina merece destaque nessa classe de fármacos, devido à maior seletividade e potência na redução dos níveis de colesterol LDL. O ácido acetilsalicílico, antiinflamatório não-esteróide, também representa uma importante alternativa terapêutica para prevenção de doenças cardiovasculares, devido à sua ampla aceitação como inibidor da agregação plaquetária. Considerando seus mecanismos de ação, estatinas e ácido acetilsalicílico podem ser usados em conjunto para a prevenção de doenças cardiovasculares. Face ao exposto e tendo-se em vista a importância dos dendrímeros como transportadores de fármacos na latenciação, o presente trabalho teve como objetivo desenvolver o pró-fármaco dendrimérico potencialmente ativo em doenças cardiovasculares contendo rosuvastatina e ácido acetisalicílico. Diversas metodologias de síntese foram realizadas na tentativa de obtenção do pró-fármaco dendrimérico composto por mio-inositol ou etilenoglicol como foco central, ácido L(-)-málico e etilenoglicol como espaçantes e rosuvastatina e ácido acetilsalicílico como compostos bioativos. Parte dos intermediários propostos foi sintetizada e purificada com sucesso. As maiores dificuldades encontradas foram a purificação dos compostos e a hidrólise seletiva da proteção do ácido málico protegido. Adicionalmente, realizaram-se estudos computacionais para prever a liberação dos fármacos do pró-fármaco dendrimérico. / Cardiovascular diseases can lead to several clinical manifestations such as myocardial infarction and stroke. Those diseases represent the main cause of death globally and this fact triggers a great interest from the pharmaceutical industries. The causes are usually related to high cholesterol levels and platelet aggregation, which are responsible for the vaso-occlusive events. Among the available drug therapy for control and prevention of cardiovascular diseases, the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), popularly known as statins, can be highlighted. Rosuvastatin deserves mention in this class of drugs due to its greater selectivity and potency in reducing the levels of LDL cholesterol. Aspirin, a nonsteroidal anti-inflammatory, also represents an important drug therapy for treatment and prevention of cardiovascular diseases, due to its widespread acceptance as a platelet aggregation inhibitor. Considering their mechanism of action, aspirin and statin can be used in association for prevention of cardiovascular diseases. This said and taken into account that dendrimers are important as carriers in prodrug design the purpose of this work was the synthesis of dendrimer prodrugs potentially active in cardiovascular diseases containing aspirin and rosuvastatin. Several synthetic methods have been used with the aim to synthesizing the dendrimer produgs composed of myo-inositol or ethyleneglycol as core, L-(-)-malic acid and ethyleneglycol as spacer groups and rosuvastatin and aspirin as bioactive compounds. Some of the proposed intermediates was synthesized and purified successfully. The main difficulties were purification of compounds and selectivy desprotection of protected malic acid. Additionally, computational studies were performed in order to predict the release of those drugs from dendrimer prodrugs

La résistance aux antibiotiques par les micro-organismes est une menace mondiale. C'est pourquoi la recherche de nouveaux médicaments revêt une importance capitale de nos jours.Le nicotinamide adénine dinucléotide (NAD) est un cofacteur essentiel et un substrat dans de nombreuses réactions biologiques. Pour cette raison, les enzymes qui l'utilisent comme substrat et celles qui participent à sa biosynthèse sont largement étudiées en tant que cibles antibactériennes potentielles. Dans ce contexte, nous nous intéressons au développement de nouveaux agents antibactériens contre la quinolinate synthase (NadA). Cette enzyme participe à la voie de biosynthèse de novo du NAD chez les procaryotes et est essentielle chez deux pathogènes : Helicobacter pylori, responsable d'ulcères et de cancers gastriques, et Mycobacterium leprae, responsable de la lèpre. Le fait que NadA n'existe que chez les procaryotes et qu'elle est essentielle chez deux agents pathogènes font de NadA une cible intéressante pour la conception de nouveaux antibactériens. La quinolinate synthase est une enzyme [4Fe-4S] où l'un des atomes de Fe est coordonné par une molécule d'eau à l'état de repos et joue le rôle d'acide de Lewis durant la catalyse. La réaction catalysée par NadA consiste en la formation d'acide quinolinique (QA), précurseur du NAD, à partir d'iminoaspartate et de dihydroxyacétone phosphate. Sur la base de la découverte dans notre laboratoire du premier inhibiteur de NadA (le DTHPA), qui agit par coordination irréversible du fer catalytique du cluster, nous avons conçu et synthétisé une famille de molécules pouvant servir d'inhibiteurs plus spécifiques de NadA. Ces molécules contiennent un cycle benzène / pyridine ou pyrazine, deux carboxylates vicinaux et un thiol en tant que groupe coordinant du fer: l’acide 4-mercaptophtalique (4MP), l’acide 6-mercaptopyridine-2, 3-dicarboxylique (6MPDC), l’acide 5-mercaptopyrazine-2, 3-dicarboxylique (5MPzDC) et l’acide 5-mercaptopyridine-2, 3-dicarboxylique (5MPDC). Nous avons démontré que ces molécules inhibent l’enzyme NadA in vitro avec un IC50 du même ordre de grandeur que celui du DTHPA (dizaine de µM). Par l’utilisation de diverses spectroscopies et de la cristallographie, nous avons démontré que l'inhibition s’effectue par la coordination de ces molécules avec le fer catalytique du cluster via leur groupement thiol. Nous avons également étudié in vitro la spécificité de ces molécules. Nous avons montré que les molécules 4MP et 5MPDC étaient des inhibiteurs spécifiques de NadA lorsqu’on les teste sur l’aconitase B, une enzyme [4Fe-4S] bactérienne, dont le cluster présente des propriétés structurales et fonctionnelles similaires à celles du cluster de NadA.Enfin, nous avons étudié l'activité d'inhibitrice de l’ensemble des molécules in cellulo sur de la voie de biosynthèse du QA chez Escherichia coli avec le DTHPA comme contrôle. Alors que les molécules 6MPDC et 5MPzDC inhibent la croissance d’E. coli de manière indépendante de la voie de biosynthèse du QA, le 4MP et le 5MPDC (les deux inhibiteurs spécifiques in vitro) n'ont montré aucune activité inhibitrice in cellulo. Ce manque d'activité pouvant être dû à un manque de pénétration des molécules à l'intérieur des bactéries, nous avons envisagé de favoriser la pénétration à l'aide d'un vecteur transmembranaire, un analogue simplifié du tétra-cyclopeptide naturel FR235222. Nous avons synthétisé et couplé le cyclopeptide à l'inhibiteur 4MP. Malheureusement, aucune inhibition de la croissance d'E. coli n'a été observée. La thèse s’est terminée en essayent de comprendre la pénétration du tétra-cyclopeptide sur bactérie, en utilisant notamment des agents fluorophores. / Resistance to antibiotics is becoming a world-wide threat. Microorganisms are able to withstand drugs leading to persistence of diseases. This is why the research of new drugs is of great importance nowadays.Nicotinamide adenine dinucleotide (NAD) is an essential cofactor and substrate in numerous biological reactions. For this reason, both the enzymes that use it as a substrate and those participating on its biosynthetic pathway are largely studied as potential antibacterial targets. In this context, we are interested in the development of new antibacterial drugs against quinolinate synthase enzyme (NadA). This enzyme participates in the prokaryotic NAD de novo biosynthetic pathway and is essential in two pathogens: Helicobacter pylori, cause of gastric ulcers and cancers, and Mycobacterium leprae, responsible of leprosy. The fact that NadA only exists in prokaryotes and the fact that it is essential for these two pathogens make it an interesting target for the design of new antibacterial drugs. Quinolinate synthase is a [4Fe-4S] cluster enzyme, where one of the Fe sites is coordinated by a water molecule in the resting state, and plays a Lewis acid role during catalysis. The reaction catalyzed by NadA consists in the formation of quinolinic acid (QA), precursor of NAD, from iminoaspartate and dihydroxyacetone phosphate. Based on the discovery in our laboratory of the first inhibitor of NadA (DTHPA) that coordinates irreversibly the catalytic iron site of the cluster, we designed and synthesized a family of molecules as potential more specific NadA inhibitors. These molecules contain a benzene/pyridine or pyrazine ring, two vicinal carboxylates and a thiol as an iron coordinating group: 4-mercaptophthalic acid (4MP), 6-mercaptopyridine-2, 3-dicarboxylic acid (6MPDC), 5-mercaptopyrazine-2, 3-dicarboxylic acid (5MPzDC) and 5-mercaptopyridine-2, 3-dicarboxylic acid (5MPDC). We demonstrated that these molecules inhibit NadA enzyme in vitro in the same range as DTHPA. Using different spectroscopies and crystallography, we demonstrated that inhibition occurs by coordination of the molecules to the catalytic iron site through their thiol group. We investigated also in vitro the specificity of these molecules. We demonstrated that 4MP and 5MPDC molecules are specific NadA inhibitors when assayed on bacterial aconitase B, a [4Fe-4S] enzyme, whose cluster displays functional and structural properties similar to those of NadA.Finally, we investigated the QA pathway inhibition activity of the four molecules in cellulo, in an Escherichia coli strain. Whereas, 6MPDC and 5MPzDC molecules inhibit E. coli growth in a QA biosynthetic pathway independent manner, 4MP and 5MPDC (the two in vitro specific inhibitors) did not show any in cellulo inhibition activity. Since this lack of activity might be due to a lack of penetration of the molecules inside bacteria, we thought about assisting the penetration of the molecules using a transmembrane carrier, a simplified analogue of the tetra-cyclopeptide FR235222 natural product. We synthetized and coupled the cyclopeptide to the 4MP inhibitor. Unfortunately, no E. coli growth inhibition was observed. The Ph.D ended by investigating the penetration of the tetra-cyclopeptide inside bacteria, using some fluorophore agents.

Cette thèse s’articule autour de systèmes innovants de délivrance d’anticancéreux pour répondre aux limitations actuelles des systèmes de type nanoparticules. Celles-ci permettent l’encapsulation d’anticancéreux pour prolonger leur temps de circulation dans le sang et diminuer leurs effets secondaires. Néanmoins les produits disponibles en cliniques ne permettent pas un contrôle précis de la libération de la substance active, ni un ciblage de la tumeur.Pour répondre à ces deux limitations, nous avons synthétisé un copolymère thermosensible ayant une température critique haute de solubilité (upper critical solution temperature, UCST) pour formuler des nanoparticules encapsulant physiquement la doxorubicine. Celles-ci permettent la libération contrôlée de la substance active par hyperthermie modérée à 43 °C. Nous avons étudié notre système d’un point de vue physico-chimique et évalué sa cytotoxicité in vitro sur des cellules de cancer de l’ovaire.Nous avons également opté pour une approche via couplage chimique entre une substance active, le paclitaxel, et le polymère afin de permettre l’administration par la voie sous-cutanée d’anticancéreux. En effet, cette voie d’administration est peu utilisée pour les anticancéreux car certains d’entre eux induisent une toxicité locale au site d’injection de type irritation / nécrose de la peau. Nous avons évalué si d’une part, l’approche prodrogue polymère hydrophile permet d’empêcher cette toxicité locale et si d’autre part, l’approche prodrogue polymère UCST permet d’obtenir des nanoparticules stables à température ambiante en vue d’une administration par la voie sous-cutanée. Une fois administrées, les nanoparticules deviennent hydrophiles par le changement de température, 34 °C dans le tissu sous-cutané, et peuvent donc diffuser librement jusqu’à atteindre la circulation sanguine. Nos travaux ont permis d’évaluer l’approche prodrogue polymère hydrophile in vivo chez la souris nude, ainsi que de décrire pour la première fois la synthèse de prodrogues polymères UCST. / This thesis focuses on innovative drug delivery systems of anticancer drugs to tackle the current limitations of formulations based on nanoparticles. These allow encapsulation of anticancer drugs to prolong their circulation time in the blood stream and to decrease side effects. Yet, nanoparticle formulations available in the clinic do not allow a precise control on the drug release nor targeting of the tumor.To overcome these hurdles, we have synthesized a thermoresponsive copolymer exhibiting an upper critical solution temperature (UCST) to formulate nanoparticles physically encapsulating doxorubicin. These allow controlled release of the anticancer drug by mild hyperthermia at 43 °C. We have studied our system from a physico-chemical point of view and evaluated its cytotoxicity in vitro on ovarian cancer cells.We have also tried a chemical coupling approach between the polymer and the anticancer drug, paclitaxel, to allow innocuous subcutaneous administration. In did, this route of administration is seldom used for anticancer drugs as some of them induce local toxicity at the site of injection in the form of skin irritation / necrosis. We assessed if a hydrophilic polymer prodrug approach allows innocuous subcutaneous administration of an irritant drug; and if a UCST polymer prodrug approach enables formation of stable nanoparticles at room temperature for subcutaneous administration. Once in the subcutaneous tissue at 34 °C, they would solubilize and become hydrophilic thus could freely diffuse to reach the blood circulation. We have managed to evaluate the hydrophilic polymer prodrug approach in vivo on nude mice and we are the first to describe the synthesis of UCST polymer prodrug.

L’adénosine est une molécule dotée d’un fort potentiel thérapeutique, mais présentant néanmoins un temps de demi-vie plasmatique extrêmement court qui limite de manière sérieuse son efficacité. Comme présenté dans l’introduction bibliographique de cette thèse, cette difficulté peut être surmontée grâce à l’utilisation de systèmes de délivrance de médicaments à base de lipides. L’adénosine peut en effet être soit encapsulée dans des liposomes, soit simplement couplée à un lipide. Parmi les « lipidizations » de l’adénosine, la « squalénisation », notamment, a favorablement modifié la biodistribution de cette substance active. Cette technique consiste à coupler l’adénosine à une molécule lipophile dérivée du squalène, générant ainsi des bioconjugués ayant la capacité de s’auto-assembler spontanément en milieu aqueux sous forme de nanoparticules d’une centaine de nanomètres de diamètre. L’injection de ces nanoparticules d’adénosine-squalène (AdSQ) par voie intraveineuse a donné des résultats très prometteurs pour le traitement de l’ischémie cérébrale et du traumatisme de la moëlle épinière. Ainsi, l’objectif de cette thèse a consisté à faire progresser le développement pré-clinique de ces nanomédicaments suivant deux axes principaux: l’étude du mécanisme d’action et l’amélioration de la formulation.De ce fait, le premier chapitre de cette thèse présente les résultats obtenus lors de l’étude in vitro du mécanisme d’action des nanoparticules d’AdSQ. Les travaux ont montré que ces nanoparticules d’AdSQ n’interagissaient pas directement avec les récepteurs à l’adénosine, mais formaient un réservoir intracellulaire d’adénosine. En effet, après internalisation, le bioconjugué d’AdSQ est clivé pour libérer l’adénosine. Celle-ci finit par être effluée par les cellules vers le milieu extracellulaire, où elle peut ainsi activer les récepteurs spécifiques situés au niveau des membranes des cellules avoisinantes. Après étude du mécanisme, l’amélioration de la formulation de ces nanoparticules a été explorée et décrite dans le deuxième chapitre. Les efforts ont été principalement concentrés sur la lyophilisation de la suspension nanoparticulaire, afin de proposer une formulation stable dans le temps et facile d’utilisation dans le cadre médical. Les conditions utilisées ont abouti au bon maintien des propriétés physico-chimiques des nanoparticules et l’obtention de solutions injectables sans risque chez l’animal. Dans son ensemble, ce travail de thèse a permis d’élargir les perspectives d’application des nanoparticules d’AdSQ grâce à une meilleure compréhension de leur mécanisme d’action ainsi que la mise au point d’une formulation plus adaptée aux besoins cliniques. / Adenosine has a high therapeutic potential but its extremely short half-life in blood seriously impairs its efficacy. As presented in the literature review, this difficulty can be overcome by using lipid-based drug delivery systems. Indeed, adenosine can be encapsulated into liposomes or conjugated to a lipid. In particular, among adenosine « lipidizations », the so-called « squalenoylation » has been shown to enhance adenosine biodistribution. This technique consists in coupling adenosine to a lipophilic squalene derivative, thus generating bioconjugates which are able to spontaneously self-assemble as nanoparticles of 100 nm of diameter in aqueous solution. Intravenous injection of these squalene-adenosine (SQAd) nanoparticles led to highly promising results for the treatment of cerebral ischemia and spinal cord injury. Thus, the aim of this thesis was to push forward the pre-clinical development of these nanomedicines following two main directions: unveiling their mechanism of action and enhancing their formulation.Thereby, the first chapter of this thesis presents the results from in vitro study on SQAd nanoparticles mechanism of action. This work has shown that SQAd nanoparticles did not interact directly with adenosine receptors, but formed an intracellular reservoir of adenosine. Indeed, after internalisation, SQAd bioconjugates acted as prodrugs by releasing free adenosine. This molecule was then efflued out of the cells into the extracellular medium, where it could activate specific membrane receptors on neighbouring cells. After studying the mechanism of action, we explored how to optimize the formulation. Results are described in the second chapter. We focus our efforts on freeze-drying the nanoparticles suspension, in order to offer a stable and easy-to-use formulation. Pre-formulation studies were conducted in order to define the optimal conditions for the preservation of nanoparticles physico-chemical properties and for an easy reconstitution of these nanoparticles suspension which can thus be safely injected intravenously. Overall, this work has widen the field of applications for SQAd nanoparticles thanks to a better understanding of their mechanism of action and the development of a formulation which is more suited to clinical needs.

Cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species and a decreased expression of enzymatic antioxidants. Prodrugs inducing additional oxidative stress can selectively induce apoptosis in cancer cells already having a high level of intrinsic oxidative stress. This study focused on the rational design and in vitro evaluation of a novel prodrug ester, (4- [(nitrooxy)methyl]phenyl-N-acetyl-L-alaninate or NPAA) activated by acylamino-acidreleasing enzyme (AARE, EC 3.4.19.1) to yield a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. NPAA shares structural features with both nitric oxide donating aspirin (NO-ASA), a wellcharacterized QM releasing anticancer prodrug, and N-acetyl-L-alanine-4-nitroanailide (AANA), a known specific substrate for AARE. AARE is a serine peptidase that is overexpressed in some tumors and cancer cell lines. The overall approach was to first predict the 3-dimensional structure of both rat (rAARE) and human AARE (hAARE) and then use the resulting low-resolution models to determine if NPAA was a plausible prodrug by estimating its affinity to hAARE and rAARE in comparison to AANA. The AARE models were constructed using a bioinformatic-based protein structure prediction webserver (I-TASSER) followed by energy minimization and refinement. The resulting models were subjected to a variety of structural quality assessments. The optimal models of hAARE and rAARE were found to have similar three-dimensional structures with a ß- propeller domain and an a/ß-hydrolase domain containing an exopeptidase catalytic site with active site residue distances typically found in serine peptidases. Protein-ligand docking studies showed that both AANA and NPAA could bind to the exopeptidase catalytic site of the hAARE and rAARE models with reasonable affinities and in a region with a highly druggable pocket. In order to validate the in silico results, NPAA was synthesized, purified, physically characterized and evaluated for its in vitro ability to deplete GSH in the presence of rAARE. As anticipated, NPAA was found to deplete GSH and this effect was completely blocked by diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, including rAARE. These studies support further efforts to optimize the design of QM releasing anticancer prodrugs targeted to AARE. Moreover, the molecular models presented here could be useful for the rational design of AARE inhibitors, which could also be exploited as potential anticancer agents.

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