De nos jours la tuberculose est une des maladies les plus meurtrières au monde. Un problème majeur est que l’agent pathogène responsable de cette maladie (Mycobacterium tuberculosis) a développé des mécanismes de résistances envers les médicaments actuels. Il devient donc urgent de trouver d’autres cibles pour développer de nouveaux antituberculeux. La biosynthèse des isoprénoïdes pourrait en être une. Les précurseurs biologiques de tous les isoprénoïdes sont l’IPP et le DMAPP qui sont synthétisés selon deux voies. La voie du mévalonate, présente chez l’Homme et la voie du méthylérythritol phosphate (MEP) laquelle est présente chez M. tuberculosis et absente chez l’homme. La fosmidomycine et la fosfoxacine, deux inhibiteurs de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de la voie du MEP ne permet pas d’inhiber la croissance de la mycobactérie. Cela est dû à l’absence de pénétration de ces composés polaires au sein de la bactérie. Pour pallier à ces problèmes de biodisponibilité, nous avons synthétisé des prodrogues lipophiles de type cycloSaligényle et arylphosphoramidate d’inhibiteurs de la DXR. Certains composés sont inhibent la croissance d’une mycobactérie non-pathogène, Mycobacterium smegmatis. / Today, tuberculosis is one of most murderous infectious diseases in the world. This disease is caused by the mycobacterium : Mycobacterium tuberculosis which is becoming more and more resistant towards antitubercular drugs. Therefore, it is urgent to find inovative targets for the development of new antitubercular drugs. The biosynthesis of isoprenoids represents such a target. The biological precursors of all isoprenoids are IPP and DMAPP which are synthesized via two pathways the mevalonate pathway, which is present in human and the methylerythritol phosphate (MEP) pathway which is present in M. tuberculosis. but absent in human. Fosmidomycin and fosfoxacine, two natural inhibitors of the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of MEP pathway, but they do not affect the growth of Mycobacterium tuberculosis cells, due to a lack of uptake of the polar drugs by the bacteria. To overcome this absence of the mycobacterial cell watll crossing of these compounds, we synthesized lipophilic cycloSaligenyl and arylphosphoramidate prodrugs of DXR inhibitors. Some compounds inhibit the growth of Mycobacterium smegmatis, a non-pathogenic model of mycobacterium.

Le développement de nouveaux antimicrobiens devient une nécessité suite à l’augmentation de souches résistantes. Les isoprénoïdes sont une cible, car ils jouent un rôle important dans le maintien des fonctions propres à chaque organisme vivant. Ils dérivent de deux précurseurs : IPP et DMAPP, synthétisés via la voie du MVA ou du MEP. Cette dernière n’étant pas présente chez l’homme mais chez de nombreuses bactéries pathogènes, toutes les enzymes de cette voie peuvent être des cibles intéressantes et notamment la désoxyxylulose phosphate réductoisomérase (DXR). Les bactéries devenant résistantes à la fosmidomycine, un inhibiteur naturel efficace de la DXR, nous avons synthétisé des analogues de cet antibiotique, fonctionnalisés judicieusement pour cibler le site du NADPH ou une poche allostérique et le site du DXP (substrat naturel). En ciblant ces deux sites, nous devrions améliorer l’affinité et l’efficacité des inhibiteurs. Les approches biligand et bisubtrat, ont été mises en œuvre. Les composés ont montré des résultats intéressants avec des IC50 dans le bas micromolaire. Un inhibiteur en particulier a montré des résultats comparables à la fosmidomycine avec un IC50 de l’ordre du nanomolaire. / The development of new antimicrobial is a necessity due to the emergence of multidrug resistance. Isoprenoids are a key target as they play an essential role in the maintenance of proper functions in all living organisms. Isoprenoids derive from two precursors: IPP and DMAPP, synthesized via the MVA or MEP pathway. The last one, absent in human but present in many pathogenic bacteria, all the enzymes of the MEP pathway therefore represent efficient targets, as the desoxyxylulose phosphate reductoisomerase (DXR). Owing the rapid emergence of resistance toward fosmidomycine, an efficient inhibitor of DXR, we synthesized analogues of this anitbiotic, appropriately functionalized to target the NADPH binding site or an allosteric pocket, as well as the DXP, the natural substrate of DXR. By targeting both sites, we should improve the affinity and efficiency of the inhibitors. The biligand and bisubstrate approaches, have been implemented. The compounds have shown interesting results, with IC50 in the low micro-molar. Notably, one inhibitor has shown comparable results to the fosmidomycine with an IC50 at nanomolar level.

Synthèse d’inhibiteurs potentiels non phosphorylés de la désoxyxylulose phosphate réductoisomérase et étude de la voie de biosynthèse des unités isopréniques chez Acanthamoeba polyphaga. De nombreux microorganismes pathogènes utilisent la voie du méthylérythritol phosphate (MEP) pour la synthèse des unités isopréniques (IPP et DMAPP). Absente chez l’homme, cette voie constitue une cible de choix pour lutter contre ces microorganismes. La fosmidomycine est un des meilleurs inhibiteurs connus à ce jour de la désoxyxylulose phosphate réductoisomérase (DXR), deuxième enzyme de cette voie. Afin d’améliorer d’une part la biodisponibilité de ce type d’inhibiteur et d’autre part de lutter contre le phénomène de résistance à cet antibiotique, nous avons synthétisé des analogues de la fosmidomycine où le groupement phosphonate est remplacé par un groupement tétrazole ou par un groupement squaryle. Les molécules synthétisées ont été testées sur la DXR d’ Escherichia coli et n’ont pas révélé d’activité inhibitrice significative.Par ailleurs, nous avons montré, par des expériences d’incorporation de glucose marqué au 13C, que l’amibe Acanthamoeba polyphaga, utilise la voie du mévalonate pour synthétiser les unités isopréniques nécessaires à la synthèse de ses stérols / Synthesis of potential non-phosphonate inhibitors of the deoxyxylulose phosphate reductoisomerase and study of the biosynthetic pathway for isoprenoids units synthesis using by Acanthamoeba polyphagaMany pathogenic microorganisms synthesize their isoprenoid units (IPP and DMAPP) via the methylerythritol phosphate pathway (MEP pathway). Absent in man, all enzymes of this metabolic route are potential targets for the design of new antimicrobials. This pathway is present in pathogenic bacteria, but absent in mammals. Hence, the development of small-molecule inhibitors for the MEP enzymes constitutes a novel approach for the design of new antimicrobials. Fosmidomycin is the most efficient inhibitor of the the deoxyxylulose phosphate reductoisomerase (DXR), the second enzyme of the MEP pathway. In an attempt to improve the pharmacological properties and the bioavailability of this antibiotic, we synthesized analogues of the fosmidomycin by replacing the phosphonate group by tetrazole or squaryl moieties. These synthesized compounds were tested on the DXR isolated from Escherichia coli.Otherwise, we showed by achieving incorporation experiments with 13C labeled glucose that the amoeba Acanthamoeba polyphaga utilize the mevalonate pathway to synthesize its sterols.

La thèse porte sur l’étude des 2 premières enzymes de la voie du MEP: la DXS et DXR. La voie du MEP conduit à la biosynthèse des isoprénoïdes chez la plupart des bactéries, dont des pathogènes. Etant absente chez l’homme, les enzymes de cette voie cible idéale pour la recherche de nouveaux antimicrobiens. L’objectif principal était d’améliorer le développement de nouveaux antimicrobiens. Nous avons utilisé des outils computationnels : méthodes de docking et de mécanique moléculaire couplée à la méthode MM/PBSA. Nous avons identifié les résidus contribuant significativement à la fixation d’un ligand dans le site actif de la DXR. Ces résultats ont été utilisés lors de la conception de nouveaux candidats inhibiteurs de type bisubstrat, biligand et difluoro phosphonate, dont 2 ont pu être synthétisés. Nous avons également développé une méthode de synthèse donnant accès au L- et D-GAP énantiomeriquement purs, dans le but d’étudier l’énantiospécificité de la DXS face à son substrat D-GAP. / This thesis concerns the study of the 2 first enzymes of the MEP pathway: DXS and DXR. The MEP pathway permits the biosynthesis of isoprénoïdes in most bacteria, including pathogenic one. As it is not present in human, enzymes of MEP pathway are effective targets in the research of new antimicrobial drugs. The objective was to advance the development of new antimicrobiotic compounds. We used computational tools: molecular docking and molecular dynamics simulations coupled with an MM/PBSA approach. We were able to identify residues that contribute significantly to the ligand binding in the DXR active site. These results were used to guide the conception of new inhibitor models, such as bisubstrates, biligands and α,α-difluoro phosphonates, two of which were synthetized. We also developed a synthesis method to obtain L- and D-GAP as enantiomerically pure molecules. The goal was to study the enantiospecificity of DXS to its substrate, D-GAP.

Submitted by biblioteca unifenas (biblioteca@unifenas.br) on 2017-09-18T20:18:02Z No. of bitstreams: 1 Roberta Soares Terra Dissertação.pdf: 5586714 bytes, checksum: b90d918d9ac7cb880dcc55847cd8c5fd (MD5) / Made available in DSpace on 2017-09-18T20:18:02Z (GMT). No. of bitstreams: 1 Roberta Soares Terra Dissertação.pdf: 5586714 bytes, checksum: b90d918d9ac7cb880dcc55847cd8c5fd (MD5) Previous issue date: 2017-03-31 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG / Salvia officinalis has been widely used in culinary and traditional medicine, and studies have identified numerous chemical compounds and potential therapeutic actions. This study evaluated the genotoxicity of lyophilized hydroalcoholic extract of S. officinalis L. (SO) leaves using the micronucleus assay in mouse bone marrow. The interaction between SO and the genotoxic effects induced by doxorubicin (DXR) was also analyzed – antigenotoxicity assay. Experimental groups consisting of male and female Swiss albinus mice (Unib: SW) were evaluated after 24-48h treatment with cyclophosphamide (CP; 50 mg/kg), DXR (5 mg/kg), NaCl 0.5-2 g/kg) and SO (0.5 g/kg) + DXR (5 mg/kg). The PCEMN analyzes showed differences (p0.05) between SO (1-2 g/kg) and NaCl treatments, regardless of gender and time. DXR induced EPCMNs significantly in both genders and treatment times. Groups of mice treated with DXR showed lower frequencies (p0.05) of PCEMNs when compared to CP control groups (50 mg/kg). Associative treatment (500 mg/kg SO + 5 mg/kg DXR) did not reduce the frequency of DXR-induced PCEMNs (p0.05). The PCE/NCE ratio between control (NaCl, CP and DXR) and experimental genotoxic and antigenotoxic (SO; SO + DXR) treatments were insignificant (p0.05). The results suggest moderately genotoxic effects (clastogeny and/or aneugeny) of S. officinalis L. leaves, dose-dependent (i.e., from 1 g/kg) and gender- and time-independent. However, S. officinalis has no systemic toxicity and antigenotoxic effects (SO + DXR) under the conditions established in the present micronucleus test in mouse bone marrow. / Salvia officinalis tem sido amplamente utilizada na culinária e na medicina tradicional, e estudos têm identificado inúmeros compostos químicos e potenciais ações terapêuticas. Esta pesquisa avaliou a genotoxicidade do extrato hidroalcoólico liofilizado de folhas de S. officinalis L. (SO) usando o ensaio do micronúcleo em medula óssea de camundongos. A interação entre SO e os efeitos genotóxicos induzidos pela doxorrubicina (DXR) também foi analisada – ensaio de antigenotoxicidade. Grupos experimentais constituídos de camundongos machos e fêmeas Swiss albinus (Unib: SW) foram avaliados após 24-48h de tratamento com ciclofosfamida (CP; 50 mg/Kg), DXR (5 mg/Kg), NaCl (150 mM), SO (0,5–2 g/Kg) e SO (0,5 g/Kg) + DXR (5 mg/Kg). As análises de EPCMNs mostraram diferenças (p0,05) entre os tratamentos de SO (1–2 g/Kg) e NaCl, independentemente do gênero e do tempo. DXR induziu EPCMNs significativamente em ambos os gêneros e tempos de tratamento. Grupos de camundongos tratados com DXR mostraram frequências menores (p<0,05) de EPCMNs quando comparados com os grupos controles CP (50 mg/Kg). O tratamento associativo (500 mg/Kg de SO + 5 mg/Kg de DXR) não reduziu a frequência de EPCMNs (p<0,05) induzida por DXR. As proporções de EPC/ENC entre tratamentos controles (NaCl, CP e DXR) e experimentais genotóxicos e antigenotóxicos (SO; SO + DXR) foram insignificantes (p0,05). Os resultados sugerem efeitos moderadamente genotóxicos (clastogenia e/ou aneugenia) de folhas de S. officinalis L., dose-dependente (i.e., a partir de 1 g/Kg) e gênero- e tempo-independentes. Contudo, S. officinalis não apresenta toxicidade sistêmica e efeitos antigenotóxicos (SO + DXR) nas condições estabelecidas no presente teste do micronúcleo em medula óssea de camundongos.

Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria. The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined. The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods. The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

The goal of this thesis is to evaluate the usability of hardware accelerated ray tracing in near-future rendering engines. Specifically, DirectX Ray Tracing API and Nvidia Turing architecture are being examined. Design and implementation of a hybrid rendering engine with support for hardware accelerated ray tracing is included and used in implementation of frequently used graphical effects -- hard and soft shadows, reflections, and Ambient Occlusion. The assessment is made in terms of difficulty of integration into a rendering engine, performance of the resulting system and suitability of implementation of chosen graphical effects. Performance parameters -- including number of rays cast per second, time to build acceleration structures and computation time on the GPU -- are tested and discussed.

La synthèse enzymatique de terpénoides chez les mammifères provient de la voie mevalonique. Récemment une voie différente a été découverte et s'est révélée être prépondérante pour de nombreux organismes comme les plantes et bactéries. L'identification d'un inhibiteur de cette cascade enzymatique permettrait le développement d'une nouvelle famille d'herbicide. Les caractéristiques de la 1-déoxy-D-xylulose 5-phosphate réductoisomérase (DXR) font de cette enzyme très spécifique une cible pour la synthèse de nouveaux composés. La Fosmidomycine ainsi que son analogue acétylé le plus proche, FR-900098 restent des références pour l'inhibition de la DXR. Dans ce contexte, l'ensemble des molécules décrites dans la littérature en tant qu'inhibiteurs a été classé en fonction des modifications apportées sur le substrat naturel ou la Fosmidomycine. A partir de l'ensemble de ces informations, cinq familles ont été synthétisées pour trouver un nouveau motif complexant. Pour deux d'entre elles, le squelette de base contient un acide phosphonique et un acide phosphinique sur lequel a été introduit la diversité moléculaire grâce aux réactions de Pudovik et de couplage pallado-catalysé. Les autres motifs complexant originaux sont constitués d'une fonction carbonyle et d'un hétérocycle en α ou β. Après optimisation de la synthèse des précurseurs, la diversité a été introduite à l'aide, par exemple, d'une réaction de trois composantes permettant la préparation d'hétérocycle. Enfin, deux modifications ont été faites sur le bras espaceur : l'introduction d'atomes de fluor pour modifier les propriétés physicochimiques ou d'un atome d'azote, point d'attache de nouveaux groupements. / The non-mevalonate pathway is widely found in higher plants and in many eubacteria, including pathogenic ones, but not in mammals. Identifying a non-mevalonate pathway inhibitor would greatly contribute to the search for new herbicides. The unique properties of 1-Deoxy-D-xylulose 5-phosphate reductoisomerase make it remarkable and rational target for drug design. The phosphonohydroxamic acid Fosmidomycin, which acts through inhibition of DXR, is a natural compound produced in the fermentation of Streptomyces and still remains, with its N-acetyl homologue FR900098, one of the most active compounds. First of all, the enzyme and all the potential inhibitors tested in literature were classified in order to understand the global quest for therapeutically useful compounds. In this context, we designed and synthesized five different families of Fosmidomycin analogues containing a new chelating unit. Two targets molecules families bearing a phosphinophonic acid as common core were imagined. Divergent approach allowed the introduction of the chemical diversity thank to powerful pallado-catalyzed coupling reaction. The other families containing carbonyl group and heterocycle in α‐ and β‐position were regarded as highly potent complexing units. Chemical diversity was introduced mainly at the end of the synthesis. For one of them convergent ring formation using three-components reaction was developed. Finally two modifications of the Fosmidomycin linker were performed by the introduction of fluorine atoms on the parent structure as well as the replacement of a carbon by a nitrogen atom in order to create a new point of modifications.

The recent emergence of a number of new viral diseases as well as the re-emergence of tuberculosis (TB), indicate an urgent need for new drugs against viral and bacterial infections. Coronavirus nsp1 has been shown to induce suppression of host gene expression and interfere with host immune response. However, the mechanism behind this is currently unknown. Here we present the first nsp1 structure from an alphacoronavirus, Transmissible gastroenteritis virus (TGEV) nsp1. Contrary to previous speculation, the TGEV nsp1 structure clearly shows that alpha- and betacoronavirus nsp1s have a common evolutionary origin. However, differences in conservation, shape and surface electrostatics indicate that the mechanism for nsp1-induced suppression of host mRNA translation is likely to be different in the alpha- and betacoronavirus genera. The Modoc virus is a neuroinvasive rodent virus with similar pathology as flavivirus encephalitis in humans. The flaviviral methyltransferase catalyses the two methylations required to complete 5´ mRNA capping, essential for mRNA stability and translation. The structure of the Modoc NS5 methyltransferase domain was determined in complex with its cofactor S-adenosyl-L-methionine. The observed methyltransferase conservation between Modoc and other flaviviral branches, indicates that it may be possible to identify drugs that target a range of flaviviruses and supports the use of Modoc virus as a model for general flaviviral studies. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is part of the methylerythritol phosphate (MEP) pathway that produces essential precursors for isoprenoid biosynthesis. This pathway is used by a number of pathogens, including Mycobacterium tuberculosis and Plasmodium falciparum, but it is not present in humans. Using a structure-based approach, we designed a number of MtDXR inhibitors, including a novel fosmidomycin-analogue that exhibited improved activity against P.falciparum in an in vitro blood cell growth assay. The approach also allowed the first design of an inhibitor that bridge both DXR substrate and co-factor binding sites, providing a stepping-stone for further optimization.

Since the discovery of Mycobacterium tuberculosis (Mtb) as the bacterial agent causing tuberculosis, the permanent eradication of this disease has proven challenging. Although a number of drugs exist for the treatment of tuberculosis, 1.7 million people still die every year from this infection. The current treatment regimen involves lengthy combination therapy with four different drugs in an effort to combat the development of resistance. However, multidrug-resistant and extensively drug-resistant strains are emerging in all parts of the world. Therefore, new drugs effective in the treatment of tuberculosis are much-needed. The work presented in this thesis was focused on the early stages of drug discovery by applying different hit identification and hit expansion strategies in the exploration of two new potential drug targets, glutamine synthetase (GS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR). A literature survey was first carried out to identify new Mtb GS inhibitors from compounds known to inhibit GS in other species. Three compounds, structurally unrelated to the typical amino acid derivatives of previously known GS inhibitors, were then discovered by virtual screening and found to be Mtb GS inhibitors, exhibiting activities in the millimolar range. Imidazo[1,2-a]pyridine analogues were also investigated as Mtb GS inhibitors. The chemical functionality, size requirements and position of the substituents in the imidazo[1,2-a]pyridine hit were investigated, and a chemical library was designed based on a focused hierarchical design of experiments approach. The X-ray structure of one of the inhibitors in complex with Mtb GS provided additional insight into the structure–activity relationships of this class of compounds. Finally, new α-arylated fosmidomycin analogues were synthesized as inhibitors of Mtb DXR, exhibiting IC50 values down to 0.8 µM. This work shows that a wide variety of aryl groups are tolerated by the enzyme. Cinnamaldehydes are important synthetic intermediates in the synthesis of fosmidomycin analogues. These were prepared by an oxidative Heck reaction from acrolein and various arylboronic acids. Electron-rich, electron-poor, heterocyclic and sterically hindered boronic acids could be employed, furnishing cinnamaldehydes in 43–92% yield.