No description available.

Opioid analgesics have been a major contribution to pain therapy with opioids being used as an effective treatment for various recalcitrant pain conditions. The drug class has come under increased scrutiny due to the raising concerns about the public health crisis of opioid misuse and addiction, thereby increasing the need for alternative and safer analgesics. The exploration of alternative pharmacotherapy for pain management has led to an increasing paradigm shift towards the development of a single-drug-multiple-target approach that takes inspiration from numerous naturally occurring drugs. The mu-opioid receptor has been the primary target for the management of pain; however, the voltage-gated sodium channel Nav1.7 is gaining attention as a putative antinociceptive target based on human genetic evidence. The proposed research aims to develop multi-target directed ligands (MTDL) that modulates two key targets for pain perception, the MOR, and Nav1.7 to generate analgesics with reduced side effects and enhanced analgesia. This will be achieved by exploiting polypharmacology to develop hybrid analgesia in two ways: (i) performing structure-activity relationship (SAR) studies to design a single drug with two pharmacophores that specifically interacts with both the targets (ii) exploiting in silico techniques by performing structure-based virtual ligand screening (VLS) of a chemical library. In our work, we report that through SAR studies and molecular docking studies that the designed compounds having in combination the pharmacophore of PZM21 and aryl sulfonamide demonstrate significant interactions between the active compounds and both the MOR and Nav1.7 proteins. This study also reports the first ever bifunctional virtual ligand screening where a library consisting of over a million compounds was screened for bifunctional activity at the MOR and the Nav1.7 ion channel. We also report the development of a novel mechanism-specific membrane potential assay to that can be used to screen for subtype selective Nav1.7 inhibitors. The research performed in this thesis will serve as a platform to explore the possibility of MTDL as potential therapeutic solutions to diseases of complex etiologies such as chronic pain. It will also serve as a starting point to exploring bifunctional VLS as a way to screen large chemical libraries for MTDLs.

Yes / The antitumor prodrug temozolomide is compromised by its dependence for activity on DNA mismatch repair (MMR) and the repair of the chemosensitive DNA lesion, O6-methylguanine (O6-MeG), by O6-methylguanine-DNA-methyltransferase (E.C. 2.1.1.63, MGMT). Tumor response is also dependent on wild-type p53. Novel 3-(2-anilinoethyl)-substituted imidazotetrazines are reported that have activity independent of MGMT, MMR, and p53. This is achieved through a switch of mechanism so that bioactivity derives from imidazotetrazine-generated arylaziridinium ions that principally modify guanine-N7 sites on DNA. Mono- and bifunctional analogues are reported, and a quantitative structure-activity relationship (QSAR) study identified the p-tolyl-substituted bifunctional congener as optimized for potency, MGMT-independence, and MMR-independence. NCI60 data show the tumor cell response is distinct from other imidazotetrazines and DNA-guanine-N7 active agents such as nitrogen mustards and cisplatin. The new imidazotetrazine compounds are promising agents for further development, and their improved in vitro activity validates the principles on which they were designed.

Worldwide, malaria is one of the deadliest diseases. In 2016 it sickened 216 million people and caused 445,000 deaths. In order to control the spread of this deadly diseases to human, we can either target the mosquito vector (Anopheles gambiae) or the parasite (Plasmodium falciparum). Due to recent emergence of resistance to current insecticides and antimalarial drugs there is a pressing need to discover and develop new agents that engage new targets in these organisms. To circumvent the effect of resistance to pyrethroid insecticides on the efficacy of insecticide treated nets (ITNs), the use of acetylcholinesterase (AChE) inhibitors on ITNs has drawn attention. In the first project, we explored a small library of γ- substituted oxoisoxazole- 2(3H)-carboxamides and isoxazol-3-yl carbamates, and nitriles as AChE inhibitors targeting wild- type (G3) and resistant (Akron) An. gambiae mosquito. In total 23 compounds were synthesized and evaluated. Both carbamates and carboximides with a 2-cyclopropylethyl side chain (1-87a and 1-88a) were extremely toxic to Akron mosquitos, yet these compounds did not exhibit appreciable selectivity between human and An. gambiae AChE. Unfortunately, none of the nitriles showed appreciable toxicity to G3 strain of the mosquitoes, nor did they inhibit An. gambiae AChE. In the second project, conducted in collaboration with Professor Michael Klemba, we focused on the mode of action of an established antimalarial drug, Mefloquine (MQ). Dr. Klemba's recently developed amino acid efflux assay was used to determine the effect of MQ and its open-ring analogs on hemoglobin endocytosis and catabolism in P. falciparum-infected erythrocytes. In total 26 MQ analogs were synthesized and 18 were studied in depth to determine their potency to inhibit leucine (Leu) efflux and parasite growth (SYBR Green). An excellent correlation (R² = 0.98) over nearly 4 log units was seen for these 18 compounds in the two assays. These data are consistent with the hypothesis that the antimalarial action of these compounds principally derives from inhibition of hemoglobin endocytosis. After this observation, a number of photo-affinity probes were designed and synthesized in hopes of isolating the molecular target of MQ. These analogs are currently being used by Dr. Klemba in pull-down experiments. In the third project, conducted in collaboration with Professor Belen Cassera, we sought to optimize a new antimalarial drug lead that would circumvent current resistance mechanisms. In Plasmodium parasites, the methylerythritol phosphate (MEP) pathway is known to be essential for its growth. This pathway is absent in humans, presenting the opportunity to develop potentially safe and effective therapeutic candidates. Previous work in the Cassera and Carlier lab had established that MMV008138 was the only compound in the Malaria Box that targeted the MEP pathway and that it was (1R,3S)-configured. My research expanded previous efforts in the Carlier group and produced synthesis of 73 analogs of MMV008138 (3-21a'1) that were tested for growth inhibition. These analogs featured variation at the A-, B-, C- and D-ring. In the process, a novel Pictet-Spengler ring expansion reaction of ortho-substituted acetphenones was discovered. The ring-expanded products were identified by means of 1D and 2D NMR experiments, HRMS, and X-ray crystallography. Among the 73 analogs prepared, four compounds showed similar growth inhibition potency to the lead 3-21a'1. In particular, the methoxyamide 3-80a, and the fluorinated A-ring analogs 3-124a, 3-124c and 3-124d all showed excellent (500-700 nM) growth IC₅₀ values against P. falciparum. All four showed full rescue upon co-application of IPP (200 μM), confirming that they target the MEP pathway. ADME-Tox evaluation of these new analogs will soon be underway. / PHD / Malaria is a severe and potentially fatal mosquito-borne disease. The continuous emergence of insecticide-resistant mosquitoes and drug-resistant parasite strains necessitates the development of novel antimalarial agents, notably those that engage new targets in these organisms. Herein we present three projects in which the synthesis and characterization of new malaria insecticide and therapeutic candidates are described. Our aim in the first project was to synthesize acetylcholinesterase (AChE) inhibitors as potential mosquitocides to be deployed on insecticide-treated nets. Three different classes of compounds were synthesized and characterized. Their potency to inhibit the wild-type and insecticide-resistant mosquito AChE, and their corresponding mosquito toxicities were assessed. Mosquito-toxic compounds were identified, but they did not show appreciable selectivity between mosquito and human AChEs. The second project was directed toward finding the biological target of a known antimalarial drug; mefloquine (MQ). Numerous different MQ analogs were synthesized, and their potency was assessed in two biochemical assays. The results of this study strongly suggest that MQ kills malaria parasites by preventing them from ingesting the red blood cell hemoglobin. The third project was concerned with the optimization of a compound (MMV008138) that kills malaria parasites by preventing it from synthesizing a key biochemical building block (IPP). Several new compounds were prepared that had similar antimalarial activity to MMV008138, of which many have better potential to serve as antimalarial drugs. In addition, these studies provided valuable insights for the design of further improved analogs.

Les résistances aux traitements actuels contre le cancer imposent de trouver de nouvelles cibles thérapeutiques. Une de ces cibles est le réseau vasculaire assurant un apport suffisant en nutriments et en oxygène à la tumeur, et permettant l’apparition de métastases. Détruire la vascularisation de la tumeur par l’utilisation d’agents antivasculaires (VDA)revient à l’asphyxier et à l’affamer, inhibant ainsi la prolifération des cellules tumorales et empêchant le processus métastatique. L’objectif de ce travail de thèse a été d’étudier des analogues contraints de l’isocombrétastatine A-4 (isoCA-4), une molécule phare du laboratoire, ayant un excellent pouvoir d’inhibition de la polymérisation de la tubuline et présentant une activité antivasculaire. Ces structures dont la double liaison est incluse dans différents types de cycles C,ont été étudiées également afin d’évaluer l’influence de l’angle dièdre formé par les noyaux A et B sur les activités biologiques des divers types de structures. Préalablement sélectionnés par modélisation moléculaire, ces analogues contraints sont de type 1-arylnaphtalène, 5-arylbenzoxépine ou 4-arylchromène et ont été préparés par des voies d’ accès originales développées dans le cadre de cette thèse. Parmi les composés synthétisés, l’analogue de type benzoxépine 3-53est aussi cytotoxique que l’isoCA-4 et possède un pouvoir d’inhibition de la polymérisation de la tubuline équivalent. Une évaluation plus poussée de son profil biologique, ainsi que celle des meilleurs représentants de chaque série chimique est actuellement en cours. / Most tumor cells rely on an efficient vascular supply for their survival, making the tumor vasculature an attractive target for anti-cancer therapy. This thesis aimed at the design and synthesis of constrained analogues of isocombretastatin A-4(isoCA-4), an antivascular agent developed in the laboratory, which exerts excellent cytotoxicities against a large panel ofcancer cell lines, and strongly inhibits tubulin polymerization. Conformationally restricted analogues of isoCA-4,featuring 1-arylnaphthalene, 5-arylbenzoxepine or 4-arylchromene skeletons were designed by computational studies andprepared by novel synthetic strategies. Of all synthesized compounds, benzoxepine analogue 3-53 strongly inhibits tubulinpolymerization and shows excellent cytotoxicities against several human cancer cell lines.

Pesquisas na área de Química Medicinal têm direcionado esforços na busca por métodos que acelerem o processo de descoberta de novos medicamentos. Dentre as diversas etapas relacionadas ao longo do processo de descoberta de substâncias bioativas está a análise das relações entre a estrutura química e a atividade biológica de compostos. Neste processo, os pesquisadores da área de Química Medicinal analisam conjuntos de dados que são caracterizados pela alta dimensionalidade e baixo número de observações. Dentro desse contexto, o presente trabalho apresenta uma abordagem computacional que visa contribuir para a análise de dados químicos e, consequentemente, a descoberta de novos medicamentos para o tratamento de doenças crônicas. As abordagens de análise exploratória de dados, utilizadas neste trabalho, combinam técnicas de redução de dimensionalidade e de agrupamento para detecção de estruturas naturais que reflitam a atividade biológica dos compostos analisados. Dentre as diversas técnicas existentes para a redução de dimensionalidade, são discutidas o escore de Fisher, a análise de componentes principais e a análise de componentes principais esparsas. Quanto aos algoritmos de aprendizado, são avaliados o k-médias, fuzzy c-médias e modelo de misturas ICA aperfeiçoado. No desenvolvimento deste trabalho foram utilizados quatro conjuntos de dados, contendo informações de substâncias bioativas, sendo que dois conjuntos foram relacionados ao tratamento da diabetes mellitus e da síndrome metabólica, o terceiro conjunto relacionado a doenças cardiovasculares e o último conjunto apresenta substâncias que podem ser utilizadas no tratamento do câncer. Nos experimentos realizados, os resultados alcançados sugerem a utilização das técnicas de redução de dimensionalidade juntamente com os algoritmos não supervisionados para a tarefa de agrupamento dos dados químicos, uma vez que nesses experimentos foi possível descrever níveis de atividade biológica dos compostos estudados. Portanto, é possível concluir que as técnicas de redução de dimensionalidade e de agrupamento podem possivelmente ser utilizadas como guias no processo de descoberta e desenvolvimento de novos compostos na área de Química Medicinal. / Researches in Medicinal Chemistry\'s area have focused on the search of methods that accelerate the process of drug discovery. Among several steps related to the process of discovery of bioactive substances there is the analysis of the relationships between chemical structure and biological activity of compounds. In this process, researchers of medicinal chemistry analyze data sets that are characterized by high dimensionality and small number of observations. Within this context, this work presents a computational approach that aims to contribute to the analysis of chemical data and, consequently, the discovery of new drugs for the treatment of chronic diseases. Approaches used in exploratory data analysis, employed in this work, combine techniques of dimensionality reduction and clustering for detecting natural structures that reflect the biological activity of the analyzed compounds. Among several existing techniques for dimensionality reduction, we have focused the Fisher\'s score, principal component analysis and sparse principal component analysis. For the clustering procedure, this study evaluated k-means, fuzzy c-means and enhanced ICA mixture model. In order to perform experiments, we used four data sets, containing information of bioactive substances. Two sets are related to the treatment of diabetes mellitus and metabolic syndrome, the third set is related to cardiovascular disease and the latter set has substances that can be used in cancer treatment. In the experiments, the obtained results suggest the use of dimensionality reduction techniques along with clustering algorithms for the task of clustering chemical data, since from these experiments, it was possible to describe different levels of biological activity of the studied compounds. Therefore, we conclude that the techniques of dimensionality reduction and clustering can be used as guides in the process of discovery and development of new compounds in the field of Medicinal Chemistry

A introdução de um grupo substituinte na molécula de um fármaco promove alterações químico-estruturais que, por sua vez, modificam suas propriedades físicoquímicas. O arranjo espacial de átomos ou grupos de átomos, em especial grupos funcionais, na molécula de um fármaco, expressos por meio de suas propriedades físico-químicas, influenciam direta ou indiretamente na interação fármaco-receptor. Esta, por sua vez, determina aspectos farmacológicos e farmacocinéticos que influem na eficácia terapêutica do medicamento. Assim, uma série de compostos 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4-fenil-substituído]-2,3-diidro-1,3,4-oxadiazolínicos foi planejada e os análogos foram sintetizados, identificados estruturalmente e avaliados in vitro quanto a atividade antimicrobiana frente a Staphylococcus aureus (cepa ATCC 25923), expressa pela determinação da concentração inibitória mínima. Cepas desta bactéria são comuns em infecções hospitalares e frequentemente apresentam caráter de multi-resistência, portanto, alternativas aos fármacos comumente empregados na terapia antibacteriana, especialmente em infecções multi-resitentes, são alvo de estudos e desenvolvimento. Relações entre mudanças estruturais de 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4- fenil-substituído]-2,3-diidro-1,3,4-oxadiazolinas e suas respectivas concentrações inibitórias mínimas podem fornecer informações sobre a influência de propriedades físico-químicas na ação antibacteriana destes compostos, informações estas que podem contribuir para o entendimento das relações entre a estrutura química e a atividade biológica desta classe de compostos, visando a identificação qualitativa e quantitativa das propriedades físico-químicas que influenciam no perfil farmacológico desta classe de compostos. Os grupos substituintes introduzidos nos derivados de 2,3-diidro-1,3,4-oxadizolinas-2,3,5-substituídas contribuem para alterações em suas propriedades físico-químicas, sendo representadas por parâmetros físico-químicos que descrevem a natureza e intensidade da alteração observada. O presente trabalho objetivou identificar, partindo das propriedades físico-químicas, fatores da estrutura química que favoreçam a atividade antimicrobiana dos compostos estudados. A atividade antimicrobiana, expressa pela concentração inibitória mínima, foi determinada pelo procedimento de microdiluição sucessiva, no qual diferentes concentrações de composto a ser analisado foram incubadas em presença de inóculo de Staphylococcus aureus, em meio de cultura líquido. As microdiluições originam concentrações decrescentes a fim de se determinar a menor concentração do composto testado onde o crescimento microbiano foi inibido. Foram realizadas comparações entre as concentrações inibitórias mínimas dos compostos analisados e destas com parâmetros estruturais que representam as propriedades físico-químicas dos compostos. Baseado nas análises comparativas, identificou-se tendências que evidenciam a preponderância de propriedades físicoquímicas sobre a atividade antimicrobiana desempenhada. Constatou-se que a hidrofobicidade influi significativamente na atividade antimicrobiana. Observou-se também que o efeito eletrônico por indução e o volume do grupo substituinte em posição para-fenila também influenciam a ação antimicrobiana, mas estas ainda não foram conclusivas, carecendo de estudos mais aprofundados que levem à compreensão dos fatores estruturais que mais influenciam a ação antimicrobiana de 2-[5-nitro-tiofen-2-il]-3-acetil-5-[4-fenilsubstituído]- 2,3-diidro-1,3,4-oxadiazolinas, de forma a fundamentar o planejamento de futuros candidatos a fármacos antimicrobianos a partir desta classe de compostos. / The introduction of a substitute group within a drug molecule promotes chemical-structure shifts, which by consequence, alters its physical-chemical properties. The atomic special design, particularly the one related to functional groups, assumed in a drug molecule, expressed by the physical-chemical properties, interferers directly or indirectly on drug-receptor interaction. Theses effects collaborate to pharmacologic and pharmacokinetics aspects of drugs, interfering with its therapeutic efficient. Therefore, a set of 2-[5-nitro-thiophen-2-yl]-3-acetyl-5-[4- substitued-phenyl]-3,4-dihydro-1,3,4-oxadiazolines compounds was designed and its analogs synthesized, structurally identified, and in vitro assayed due to its antimicrobial activity against ATCC 25923 Staphylococcus aureus strains, through the determination of minimum inhibitory concentration. Strains of such bacterial species are commonly present within hospital infections, and frequently appear as multi-resistant variant. Consequently, alternatives to the agents usually applied in antimicrobial chemotherapy, particularly to multi-resistant infections, are target for drug research and development. Relations among structural shifts on 2-[5-nitro-thiophen-2-yl]-3-acetyl-5-[4-substitued-phenyl]-3,4-dihydro-1,3,4-oxadiazolines compounds and its respective minimum inhibitory concentration may lead to information about the influence of physical-chemical properties in antimicrobial activity. Continually, such information might contribute to the elucidation of chemical structure-biological activity relationship of these compounds. The substitutes groups inserted on 2,3,5-substituted-3,4-dihydro-1,3,4-oxadiazolines derivates contribute to differs the molecule physical-chemical properties, which is represented by its physical-chemical parameters, which describe the nature and intensity of the observed modification. Therefore, the following study aimed to identify, throughout physical-chemical properties, chemical structure factors that favor the antimicrobial activity of the synthesized compounds. The antibacterial activity, expressed as the minimum inhibitory concentration, was quantified by the microdilution method, where gradual concentrations of the tested conpound were incubated within ATCC 25923 Staphylococcus aureus cells suspended in broth medium. The microdilutions originated descending concentrations, in order to spot the minimum concentration whose microbial growth was inhibited. The minimum inhibitory concentration regarded to each compound was correlated to its physical-chemical parameters. Based on observed relations, it could be identified evidences assuming the physical-chemical properties relevance on antimicrobial assay. It was verified that hydrophobic effects interferes with antimicrobial activity, as well as inductive electronic effects and molecular volume of substitute group in para-phenyl position. These factors contribute to physical-chemical properties shifts and therefore play a role on the antimicrobial action. However, these latest two influences were enable to conclude, suggesting the need of more extensive studies. So that, a more profound comprehension about structure factors that interfere with 2,3,5-substitued-3,4-dihydro-1,3,4-oxadiazolines derivates might lead to the rational design of potential antimicrobial agents among this class of compounds.

Pesquisas na área de Química Medicinal têm direcionado esforços na busca por métodos que acelerem o processo de descoberta de novos medicamentos. Dentre as diversas etapas relacionadas ao longo do processo de descoberta de substâncias bioativas está a análise das relações entre a estrutura química e a atividade biológica de compostos. Neste processo, os pesquisadores da área de Química Medicinal analisam conjuntos de dados que são caracterizados pela alta dimensionalidade e baixo número de observações. Dentro desse contexto, o presente trabalho apresenta uma abordagem computacional que visa contribuir para a análise de dados químicos e, consequentemente, a descoberta de novos medicamentos para o tratamento de doenças crônicas. As abordagens de análise exploratória de dados, utilizadas neste trabalho, combinam técnicas de redução de dimensionalidade e de agrupamento para detecção de estruturas naturais que reflitam a atividade biológica dos compostos analisados. Dentre as diversas técnicas existentes para a redução de dimensionalidade, são discutidas o escore de Fisher, a análise de componentes principais e a análise de componentes principais esparsas. Quanto aos algoritmos de aprendizado, são avaliados o k-médias, fuzzy c-médias e modelo de misturas ICA aperfeiçoado. No desenvolvimento deste trabalho foram utilizados quatro conjuntos de dados, contendo informações de substâncias bioativas, sendo que dois conjuntos foram relacionados ao tratamento da diabetes mellitus e da síndrome metabólica, o terceiro conjunto relacionado a doenças cardiovasculares e o último conjunto apresenta substâncias que podem ser utilizadas no tratamento do câncer. Nos experimentos realizados, os resultados alcançados sugerem a utilização das técnicas de redução de dimensionalidade juntamente com os algoritmos não supervisionados para a tarefa de agrupamento dos dados químicos, uma vez que nesses experimentos foi possível descrever níveis de atividade biológica dos compostos estudados. Portanto, é possível concluir que as técnicas de redução de dimensionalidade e de agrupamento podem possivelmente ser utilizadas como guias no processo de descoberta e desenvolvimento de novos compostos na área de Química Medicinal. / Researches in Medicinal Chemistry\'s area have focused on the search of methods that accelerate the process of drug discovery. Among several steps related to the process of discovery of bioactive substances there is the analysis of the relationships between chemical structure and biological activity of compounds. In this process, researchers of medicinal chemistry analyze data sets that are characterized by high dimensionality and small number of observations. Within this context, this work presents a computational approach that aims to contribute to the analysis of chemical data and, consequently, the discovery of new drugs for the treatment of chronic diseases. Approaches used in exploratory data analysis, employed in this work, combine techniques of dimensionality reduction and clustering for detecting natural structures that reflect the biological activity of the analyzed compounds. Among several existing techniques for dimensionality reduction, we have focused the Fisher\'s score, principal component analysis and sparse principal component analysis. For the clustering procedure, this study evaluated k-means, fuzzy c-means and enhanced ICA mixture model. In order to perform experiments, we used four data sets, containing information of bioactive substances. Two sets are related to the treatment of diabetes mellitus and metabolic syndrome, the third set is related to cardiovascular disease and the latter set has substances that can be used in cancer treatment. In the experiments, the obtained results suggest the use of dimensionality reduction techniques along with clustering algorithms for the task of clustering chemical data, since from these experiments, it was possible to describe different levels of biological activity of the studied compounds. Therefore, we conclude that the techniques of dimensionality reduction and clustering can be used as guides in the process of discovery and development of new compounds in the field of Medicinal Chemistry

La transglutaminase tissulaire (TG2) est une enzyme de la famille des transglutaminases (EC 2.3.2.13) qui est exprimée de manière ubiquitaire chez les mammifères. Cette enzyme catalyse la formation d'une liaison amide intra- ou intermoléculaire entre un résidu glutamine et un résidu lysine. Ce processus biologique conduit à la modification post-traductionnelle des protéines. Un nombre croissant de publications associe la surexpression de cette enzyme et la déréglementation de son activité, avec un certain nombre de pathologiques humaines telles que les maladies neurodégénératives (maladie d’Alzheimer, maladie de Huntington, maladie de Parkinson), la fibrose tissulaire, certains cancers et la maladie cœliaque. Le développement d'inhibiteurs puissants et sélectifs de la TG2 est primordial pour identifier soit des outils pharmacologiques pour comprendre les processus biologiques dépendant de cette enzyme ou soit des candidats médicaments pour traiter les pathologies liées à la surexpression de la TG2. La majorité des composés inhibiteurs synthétisés jusqu'à présent agissent en bloquant de manière irréversible la réaction de transamidification de la TG2 en ciblant spécifiquement la cystéine 277 présente dans le site actif de la TG2.L’objectif de ce travail a été d’identifier et de sélectionner des molécules de faible poids moléculaire inhibant de façon sélective et puissante l’activité de transamidification de la TG2. Nous présenterons l’optimisation de deux séries originales de composés (synthèse, études de relation de structure-activité) comportant un noyau aromatique central de type naphtalénique ou indolique et une fonction acrylamide comme accepteur de Michael pour piéger la fonction thiol de la cystéine 277. Un certain nombre de composés synthétisés montre une inhibition nanomolaire de la TG2 (IC50 = 1.7-6 nM) avec un excellent profil de sélectivité vis-à-vis de TG1, TG6 et FXIIIa (IC50 > 10 µM). Ces inhibiteurs inhibent efficacement la TG2 dans des extraits de tissus et de cellules. Aucune toxicité apparente n’a été observée pour des concentrations inférieures à 10 µM d’inhibiteur sur les lignées vSMCs et SH-SY5Y. Les valeurs de KI, kinact et kinact/KI ont été également déterminés sur deux inhibiteurs sélectionnés (23b et 78f) pour leurs activités biologiques. La formation d’une liaison covalente entre la cystéine 277 de la TG2 et ces deux inhibiteurs a été prouvée par digestion trypsique suivie d’une analyse LC-MS/MS / Tissue transglutaminase (TG2) is a ubiquitously expressed enzyme of the mammalian transglutaminase (TG) family which catalyzes the formation of an intra- or inter-molecular isopeptide bond between a glutamine and a lysine, leading to the post-translational modification of proteins. An increasing number of literature has associated the over-expression of this enzyme, and the deregulation of its activity, with a number of human physio-pathological states like neurodegenerative disorders (Alzheimer’s disease, Huntington’s disease, Parkinson’s disease), tissue fibrosis, certain cancers, and celiac disease. The development of potent and selective TG2 inhibitors has become primordial to reach either a pharmacological probe, to understand the biological processes that depend on this enzyme, or a drug candidate, to treat the pathologies related with its overexpression. The majority of the inhibitory compounds synthesized so far act by irreversibly blocking the transamidation reaction of TG2. These TG2 inhibitors specifically target the cysteine 277 present in the TG2 active site. The aim of this work was the identification and selection of new potent and selective small molecules to inhibit the TG2 transamidation activity. We present the optimization of two new series of compounds (synthesis, structure-activity relationship studies) bearing naphthalene or indole aromatic rings as the central backbone structure. Both series present an acrylamide group as the Michael acceptor in order to react with the thiol group of cysteine 277. Several of the synthesized compounds showed a nanomolar inhibition over TG2 (1.7-6 nM) with an excellent selectivity profile over TG1, TG6 and FXIIIa (IC50 > 10 µM). These inhibitors showed high specificity on inhibiting TG2 in tissue and cell extracts. No apparent toxicity up to 10 µM was observed in vSMCs and SH-SY5Y cell lines. Their KI, kinact et kinact/KI were also determined on two selected inhibitors (23b and 78f) for their biological activities. The formation of a covalent bond between the cysteine 277 of TG2 and these two inhibitors was proven by tryptic digestion followed by LC-MS/MS analysis

Thromboxane A2 (TXA2) is an important mediator metabolized from arachidonic acid through the cyclooxygenase pathway, mainly in platelets and macrophages. It is a potent inducer of platelet aggregation and smooth muscle contraction. Its overproduction has been detected in pathologies such as stroke, asthma, myocardial infarction or atherosclerosis. The action of TXA2 is mediated by a specific G-protein coupled receptor (TP) of which two alternative spliced isoforms, TPalpha and TPbeta, have been described. The exact role of these two isoforms is not clearly understood. However, recent studies have described their implications in vascular physiology and pathology. The inhibition of the action of TXA2 on platelets and blood vessels would be interesting as original therapies against cardiovascular diseases. Consequently, the design of TP receptor antagonists remains of great interest in cardiovascular medicine. In the laboratory of medicinal chemistry (University of Liège, Belgium), several nitrobenzenesulfonylureas, derived from torasemide (a loop diuretic), have been previously described as TP receptor antagonists. Two compounds, BM573 and BM613 were among the most interesting molecules identified in that previous work. The present project is divided in two parts. First, we have determined the pharmacological properties of BM573 and BM613 as thromboxane synthase inhibitors and TP receptor antagonists, in vitro and in vivo. In our assays, these two compounds were proved to have high affinity for both TPalpha and TPbeta, to be potent antiplatelet agents, to inhibit thromboxane synthase and TP-mediated smooth muscle contraction. Additionally, they significantly reduced the size of the thrombus in a rat model of ferric chloride-induced arterial thrombosis. Consequently, we demonstrated that the TP receptor antagonists BM573 and BM613, belonging to the chemical family of nitrobenzenesulfonylureas, could be regarded as antiplatelet and antithrombotic agents potentially useful in thromboxane-related diseases such as stroke or myocardial infarction. Secondly, given the interesting pharmacological profile of BM573 and BM613, we have designed and synthesized several series of compounds derived from these two agents. We have evaluated the binding properties (affinity) of the first generation (+/- 35 original derivatives) of compounds on either TPalpha or TPbeta, transiently expressed in COS-7 cell lines. Additionally, we have measured the ability of our drugs to inhibit the intracellular calcium mobilization upon TPalpha or TPbeta stimulation. To confirm our results, we also assessed the antiplatelet properties of our drugs by means of determination of inhibition of human platelet aggregation. On the basis of the results obtained with these in vitro assays, we have synthesized and evaluated a second generation of derivatives (+/- 35 original compounds) and improved the selectivity of several original compounds for TP receptor isoforms. The originality of this work was to evaluate a large library of synthetic compounds on both TP receptor isoforms, using specific pharmacological tests. By means of structure-activity relationship studies, we were able to identify chemical groups implicated in selectivity and to propose lead compounds for development of highly specific TPalpha or TPbeta antagonists. Besides, we have identified an in vivo drug candidates for prevention of thrombosis and pathological platelet aggregation./Le thromboxane A2 (TXA2) est un métabolite de la cascade de lacide arachidonique (AA) par la voie des cyclooxygénases et de la thromboxane synthase, principalement formé dans les plaquettes et les macrophages. Le TXA2 est un puissant inducteur de lagrégation plaquettaire et de la contraction des muscles lisses vasculaires et bronchiques. Par ailleurs, une augmentation des taux en TXA2 a été constatée dans différentes pathologies : l'infarctus du myocarde, l'atherosclérose, les accidents vasculaires cérébraux, ou encore l'asthme. Laction du TXA2 sur les tissus résulte de la stimulation dun récepteur appartenant à la famille des récepteurs couplés aux protéines G. Ce récepteur au TXA2 (TP) présente deux isoformes générées par épissage alternatif, TPalpha et TPbeta. Le rôle physiologique exact de ces deux isoformes n'est pas encore connu. Cependant, de récents travaux ont mis en évidence leur importance, notamment dans la physiologie vasculaire et dans certaines pathologies. Linhibition de laction du TXA2 au niveau des plaquettes et des vaisseaux sanguins pourrait donc être une stratégie thérapeutique innovante pour traiter et prévenir les maladies cardiovasculaires. En conséquence, le développement dantagonistes des récepteurs TP reste dun grand intérêt en médecine cardiovasculaire. Des études de pharmacomodulation avaient permis au Laboratoire de Chimie Pharmaceutique (Université de Liège, Belgique) d'identifier des nitrobenzènesulfonylurées, dérivées du torasémide (un diurétique de lanse), présentant un puissant antagonisme des récepteurs TP. Parmi ceux-ci, deux composés, le BM573 et le BM613, faisaient parties des molécules les plus intéressantes identifiées au cours de ces précédentes recherches. Ce projet est divisé en deux parties. Premièrement, nous avons déterminé les propriétés pharmacologiques du BM573 et du BM613 en tant quinhibiteurs de la thromboxane synthase et antagonistes des récepteurs TP, in vitro et in vivo. Au cours de nos expériences, ces deux composés se sont révélés posséder une grande affinité pour TPalpha et TPbeta, être de puissants agents antiplaquettaires, des inhibiteurs de la thromboxane synthase et de la contraction des muscles lisses induite par le TXA2. En outre, lutilisation de ces produits dans un modèle de thrombose artérielle induite par le chlorure ferrique chez le rat a provoqué une réduction significative du thrombus formé. En conséquence, nous avons démontré que le BM573 et le BM613, appartenant à la famille chimique des nitrobenzenesulfonylurées, pouvaient être considérés comme des agents antiplaquettaires et antithrombotiques, potentiellement utiles en tant quagents thérapeutiques dans des pathologies associées au TXA2 telles que linfarctus du myocarde ou laccident vasculaire cérébral. Ensuite, nous nous sommes concentrés sur l'activité de cette famille de composés (les nitrobenzènesulfonylurées) vis-à-vis des deux isoformes du récepteur au thromboxane. Pour ce faire, nous avons conçu et synthétisé de nombreuses séries de composés dérivés du BM573 et du BM613. Nous avons tout dabord évalué laffinité de la première génération de composés (+/- 35 dérivés) sur des lignées cellulaires (COS-7) exprimant sélectivement soit TPalpha soit TPbeta. De plus, nous avons mesuré la capacité de ces composés à inhiber la mobilisation de calcium intracellulaire ([Ca2+]i) induite par la stimulation des deux isoformes TPalpha et TPbeta séparément. Nos résultats ont été confirmés sur agrégation plaquettaire humaine. Sur la base des résultats obtenus avec cette première génération de produits, nous avons synthétisé une seconde génération (+/- 35 dérivés) de composés, et avons réussi à augmenter la sélectivité en faveur de TPbeta pour certains produits. Loriginalité de ce travail réside dans le fait que nous avons évalué un nombre de produits importants sur TPalpha et TPbeta, au moyen de tests pharmacologiques spécifiques. Grâce à des études de relation structure-activité, nous avons identifié des groupements chimiques impliqués dans la sélectivité entre les deux isoformes. Nous pouvons donc proposer des structures "chef de file" pouvant être utiles pour le développement de composés hautement sélectifs, soit pour TPalpha, soit pour TPbeta. Par ailleurs, nous avons identifié in vivo des candidats pour le développement dagents thérapeutiques pour la prévention des thromboses et des autres pathologies provoquées par une activation plaquettaires excessive.