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N-linked glycosylation is the most common form of post-translational modification in nature and is essential to almost all enveloped viruses, including members of the Flaviviridae family. The host cell N-linked glycoprotein processing pathway is utilised by these viruses and as such has long been identified as a potential target for the development of antiviral drugs. Here, the antiviral mechanisms of three classes of small molecules targeting the secretory pathway and altering viral envelope glycosylation are investigated, using the HCV surrogate model, BVDV. The antiviral activity of imino sugars, principally through α-glucosidase inhibition, is well-characterised and here, a group of novel adamantyl coupled imino sugars are investigated and demonstrated to inhibit ER α glucosidases, which correlates with their antiviral activity against BVDV. Additionally, BVDV is used to study the antiviral mechanism of action of nitazoxanide. Nitazoxanide, the parent compound of the thiazolide class of structures, is a broadly antimicrobial compound with antiviral activity against HBV, HCV, influenza, JEV and others. Here, nitazoxanide is shown to be antiviral against BVDV by inducing Ca²⁺ release from ATP-sensitive intracellular calcium stores, disrupting ER-Golgi trafficking and inhibiting complex glycan formation. Finally, the potential of Golgi endo-α-mannosidase as an antiviral target is explored, using the endomannosidase inhibitor glucose-isofagomine in conjunction with the imino sugar α-glucosidase inhibitor NAP-DNJ. Endomannosidase is shown to be a valid antiviral target for BVDV, both alone and in combination with α-glucosidase inhibition, and is utilised by viral glycoproteins to acquire complex glycan structure, even in the absence of α-glucosidase inhibition. Altogether, this work furthers our understanding of the varied antiviral mechanisms of small molecules targeting the secretory pathway, enhancing the search for novel antiviral drugs directed against host cell machinery.

Dans ce travail, nous avons étudié la contribution que les inhibiteurs de glicosidases ont dans la réponse de défense du blé à Fusarium graminearum. Nous avons démontré que les inhibiteurs de xylanases ont la capacité à la fois de contenir l'activité de dégradation de xylanases sécrétées par l'agent pathogène, et de limiter la possibilité de provoquer nécrose dans les tissus du blé. Nous avons démontré que l’expression de la PvPGIP2 dans la lemme, la paléa, les anthères et le rachis détermine une réduction des symptômes de la fusariose de l’épi, au même niveau de l’expression constitutive de cet inhibiteur. Inversement, l'expression de la PvPGIP2 dans l'endosperme ne détermine pas une réduction des symptômes de la maladie. Cela indique que, lorsque l'agent pathogène a atteint ce tissu, l'activité de polygalacturonases de l'agent pathogène n’est pas indispensable pour la propagation fongique. Enfin, la combinaison des différents inhibiteurs de glicosidases, qui renforcent différentes parties de la paroi cellulaire dans le même génotype, a été efficace pour réduire les symptômes de la fusariose, par rapport aux génotypes qui présentent seulment un type d’inhibiteur. Nous avons démontré cet aspect à travers les plantes qui expriment la PvPGIP2 et le TAXI-III. Au contraire, les génotypes qui expriment la PvPGIP2 et l’AcPMEI n’ont pas montré un effect additif sur la résistance, probablement parce que ils renforcent la même partie de la paroi cellulaire, c’est-à-dire la pectine. / In this work we studied the contribution of glycosidase inhibitors in the defense response of wheat against Fusarium graminearum. We demonstrated that xylanase inhibitors are able to limit both the degrading activity of the xylanases secreted by the pathogen and to limit their ability to induce necrosis in wheat cell suspensions and tissues.We demonstrated that the expression of PvPGIP2 in lemma, palea, anthers and rachis causes a reduction in Fusarium head blight symptoms, at the same level of the constitutive expression of this inhibitor. On the contrary, the expression of PvPGIP2 in the endosperm did not result in a reduction of disease symptoms, suggesting that once the pathogen has reached the endosperm, the activity of polygalacturonases secreted by the pathogen is not essential for the progression of symptoms.The pyramiding of glycosidase inhibitors in the same genotype is effective in reducing FHB symptoms although it depends on the specific combination. Pyramiding of PvPGIP2 and AcPMEI does not enhance further wheat resistance against FHB, possibly because they target the same virulent component secreted by the pathogen, that is PG. Conversely, the pyramiding PvPGIP2 and TAXI-III supports a further improvement of resistance compared to plants carrying only PvPGIP2 or TAXI-III.

Récemment, le concept de chaperon pharmacologique a émergé pour le traitement des maladies lysosomales. Comme inhibiteurs réversibles de glycosidases mutantes impliquées dans ces maladies, les chaperons pharmacologiques sont capables, à des concentrations sub-inhibitrices, de sauver ces enzymes des mécanismes de destruction du réticulum endoplasmique (RE). Ainsi, une partie de l’activité enzymatique est restaurée. Les iminosucres sont connus pour être une classe importante de chaperons pharmacologiques. Au cours de ce travail de thèse, de nouvelles classes d’iminosucres mono- et multivalents ont été conçues et synthétisées. Nos objectifs étaient de mettre en évidence de nouveaux chaperons pour la β-glucocérébrosidase, impliquée dans la maladie de Gaucher, mais également d’identifier de nouveaux inhibiteurs des α-glucosidases du RE impliquées dans la destruction de la protéine déficiente chez les malades atteints de la mucoviscidose. Plusieurs stratégies ont été mises en œuvre: l’utilisation d’une méthodologie de diamination d’alcènes pallado-catalysée, d’une méthodologie permettant la synthèse rapide d’une bibliothèque de composés iminosucres par chimie click ou encore de la multivalence. Une étude poussée sur la multivalence et l’inhibition de glycosidases a également été réalisée en faisant varier des paramètres clés de la multivalence tels que la valence, la charpente, le linker, ou encore la nature des ligands iminosucres. Le premier exemple d’un effet multivalent puissant jusqu’à quatre ordre de grandeur sur l’inhibition de glycosidases a été mis en évidence avec des systèmes iminosucres multivalents basés sur des charpentes de type β-cyclodextrine et fullerène C60. / Recently an innovative concept for the treatment of lysosomal diseases as emerged called pharmacological chaperone. Pharmacological chaperones are reversible inhibitors of the deficient glycosidases involved in these diseases. These molecules are able, at sub-inhibitory concentrations, to stabilize the enzymes and rescue them from the destruction by the quality control system of the endoplasmic reticulum. A part of the catalytic activity of the enzyme could be restored. Iminosugars are known to be an important class of pharmaceutical chaperones. During this PhD work, novel classes of mono- and multivalent iminosugars were designed and synthesized in order to identify novel pharmacological chaperones for the glycosidase: β-glucocerebrosidase involved in Gaucher’s disease and novel inhibitors of the α-glucosidases involved in the destruction of the defective protein delF508CFTR in cystic fibrosis. Several strategies were applied to achieve this aim. These strategies consist in the use of a synthetic methodology of palladium catalyzed alkenes diamination, the use of an efficient methodology to synthesize a library of novel iminosugars by click chemistry and the use of multivalency. A full study on the impact of multivalency on glycosidases inhibition was also completed by changing crucial structural parameters including valency, scaffold, linker and ligand. The first strong multivalent effect on glycosidases inhibition up to four orders of magnitude was reported with multivalent iminosugars based on β-cyclodextrin or C60 fullerene cores.

Notre équipe a récemment décrit la synthèse de nouveaux iminosucres de type indolizidines et quinolizidines, qui se sont avérés être de puissants inhibiteurs sélectifs d’α-glucosidases (IC50 nanomolaires). Ces molécules, de structure originale, ont la particularité de posséder un centre quaternaire en jonction de cycles et une configuration D-gluco. Dans le but de mieux comprendre le mode d’interaction de ces molécules avec les glycosidases, nous nous sommes intéressés à la synthèse de leurs analogues comportant un motif aziridine, inclus dans un squelette 1 azabicyclo[4.1.0]heptane.L’approche synthétique que nous avons choisie pour accéder aux aziridinyl iminosucres implique une cycloaddition 1,3-dipolaire régio- et stéréoselective de cétonitrones dérivées de sucres avec des alcynes pour former des isoxazolines et un réarrangement de Baldwin stéréosélectif en 2 acylaziridines. Pour les acylaziridines polybenzylées la conversion en aziridinyl iminosucres a été effectuée par réduction diastéréosélective de leur fonction cétone et déprotection dans des conditions réductrices de Birch. Pour les acylaziridines polyacétylées les iminosucres finaux ont été obtenus par réduction en présence d’hydrures métalliques.Grâce à cette stratégie de synthèse originale, efficace, et économique en atomes, nous avons préparés 7 aziridinyl iminosucres, et 5 isoxazolines polyhydroxylées. Tous ces nouveaux iminosucres ont été évalués comme inhibiteurs de glycosidases, et se sont avérées être des inhibiteurs moyens (IC50 micromolaires) mais sélectifs des α-glucosidases. L’obtention de complexes cristallins avec une α-glucosidase bactérienne a permis de montrer par cristallographie leur mode d’interaction avec cette enzyme, à l’échelle moléculaire. / Our team has recently described the synthesis of new indolizidine and quinolizidine iminosugars, which revealed to be potent and selective α-glucosidase inhibitors (nanomolar IC50). These original molecules exhibit a quaternary center at the ring junction and a D-gluco configuration. With the aim to define the mode of interaction of these molecules with glycosidases, we engaged in the synthesis of analogues containing an aziridine moiety included in a 1-azabicyclo[4.1.0]heptane scaffold.Our synthetic approach towards aziridinyl iminosugars involves a stereoselective 1,3-dipolar cycloaddition between carbohydrate-derived cyclic ketonitrones and alkynes, and a stereoselective Baldwin rearrangement into 2-acylaziridines. The latter (if polybenzylated) were converted into aziridinyl iminosugars through diastereoselective reduction of their ketone function and deprotection under Birch conditions. In the case of polyacetylated acylaziridines, the final iminosugars were obtained by reduction with hydrides.Using this strategy, we have prepared 7 aziridinyl iminosugars, and 5 polyhydroxylated isoxazolines. All these new iminosugars were evaluated as glycosidase inhibitors, and they revealed to be modest (micromolar IC50) but selective inhibitors of α-glucosidases. Their complexes with a crystalline bacterial α-glucosidase allowed crystallographic elucidation of their mode of interaction with this enzyme at the molecular level.

Glycosidases are carbohydrate processing essential enzymes necessary for the growth and development of all organisms such as intestinal digestion, post-translational processing of glycoproteins and the lysosomal catabolism of glycoconjugates. The function of these glycosidases is limited and studies are still in progress to understand their function at cellular level. In recent years, biological role of carbohydrates has resulted in various carbohydrate-based therapeutics2. These carbohydrates serve as a tool to study the function of glycosidases by inhibiting their active site. The concept of inhibition is yet another approach for the discovery of drugs. Glycosidase inhibitors studied are often sugar analogs and a wide range of such inhibitors are reported in the literature.3, 4 Thiosugars, in particular, have gained new perspectives owing to their electronic, geometric, conformational and flexibility differences, as sulfide moiety being less electronegative and more polarizable than the oxygen counter-part.5 These differences make the thiosugars distinct from their oxygen analogs and hence can mimic the active site of the enzyme. Many molecules are reported to be promising glycosidase inhibitors but are not easily accessible due to difficulties in their synthesis. Hence, the chemical synthesis of thio-analogs of carbohydrates, by synthetic routes, remains a major challenge. To address the complexity of synthesis and to make available new strategies, we envisioned the use of benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS4, a versatile and efficient sulfur transfer reagent. Objectives of the study: a. Design novel thiosugars as glycosidase inhibitors. b. Devise strategy for the synthesis of novel thiosugars through a simple, practical approach. c. Evaluate the synthesized molecules as glycosidase and HIV-1 protease inhibitors, in silico. d. Study miscellaneous applications of the novel thiosugar-derived thialactones. The thesis is divided into five sections: Section A entitled “Synthesis of deoxythiosugars and thiosugar-based lactones” is divided into two parts, Part A and Part B. Part A – “An introduction and background on thiosugars and sulfur transfer reagents” has been provided. A brief discussion of sulfur transfer reagents in carbohydrate synthesis and earlier work related to the use of benzyltriethylammonium tetrathiomolybdate, [BnEt3N]2MoS4, as an efficient sulfur transfer reagent have been provided. Part B –“Design of inhibitors of glycosidases and HIV-1 protease” deals with the design of inhibitors of glycosidase and HIV-1 protease. The designed thiosugar molecules exhibit the characteristics of sugars and will act as planar molecules to mimic the active site conformation of a good inhibitor. Synthetic methodologies devised and adopted for the synthesis of constrained sugar-derived thialactones include: (a) Double displacement, (b) Displacement-cum-intramolecular thia-Michael addition, (c) Epoxide ring-opening-cum-intramolecular thia-Michael addition, and (d) Displacement-cum-epoxide ring opening in an intramolecular fashion. In all the above mentioned strategies, sulfur transfer step is the crucial step which was achieved by the use of benzyltriethylammonium tetrathiomolybdate [BnEt3N]2MoS46 as the key reagent. (a) Various constrained thialactones synthesized by double displacement strategy using tetrathiomolybdate as the sulfur transfer reagent are shown in Scheme – 1. (b) A number of constrained thialactones were synthesized following nucleophilic displacement-cum-intramolecular thia-Michael addition strategy as shown in Scheme – 2. (c) Synthesis of bicyclic thiolactones was achieved using the strategy of epoxide ring-opening-cum-intramolecular thia-Michael addition. (Scheme – 3) (d) A few bicyclic thialactones were synthesized through displacement-epoxide ring opening-cyclization as shown in Scheme – 4. The methodology was also utilized for the synthesis of thiosugar derivatives and azido-thialactones. (Fig. 1) Figure 1 Synthesis of deoxythiosugars: The bicyclic thialactones (designed as inhibitors) on reduction with borohydride exchange resin (BER) easily furnished the deoxythiosugars (Fig. 2). It is worth mentioning that the synthesis of these thiosugars as reported in the literature involved lengthy procedures whereas the present methodology turns out to be short and concise. Figure 2 Section B entitled “Synthesis of amines, β-amino acids and novel thiosugar-based dehydroamino acids” comprises a brief introduction on the importance of amines, β-amino acids and dehyroamino acids. In this section the effective utilization of benzyltriethylammonium tetrathiomolybdate as a key reagent for reductive transformations and its application in the synthesis of amines, β-amino acids and dehyroamino acids have been presented. A one pot reduction of azides to amines followed by intermolecular aza-Michael addition employing tetrathiomolybdate was achieved to furnish a number of different β-amino esters as shown in Scheme -4: Scheme 4 The study was further extended to the reduction of a few anomeric azides to afford the corresponding anomeric amines and derivatives. (Fig. 3) Figure 3 A one-pot thia-Michael addition-vinyl azide reduction in a tandem fashion employing benzyltriethylammonium tetrathiomolybdate was studied and was shown to be effective for the synthesis of thiosugar derived dehydroamino acid derivatives. (Scheme – 5) Scheme 5 Section C entitled “Molecular docking studies of deoxythiosugar probes” gives an overview of different glycosidases, HIV-1 protease and their inhibitors. This section also deals with a brief introduction on active site conformations of potent inhibitors. In this connection we have studied the crystal conformations of the synthesized molecules whose conformations were the same as that of the existing inhibitors in the active site. (Fig. 4) With this background in silico study of the synthesized deoxythiosugar probes was conducted on human glycosidases: α-mannosidase, α-galactosidase, β-glucosidase and HIV-1 protease, respectively. Figure 4 Molecular docking was carried out using Autodock suite, molecular modeling simulation. Separate docking procedures were employed for the four different receptors. The PDBs representing the four enzyme targets were 2V3D, 3H53, 1X9D and 3I8W for β–glucosidase, α–galactosidase, α–mannosidase and HIV–1 protease respectively. The control compounds used for α–mannosidase were mannostatin and kifunensine. NMB, THK, and BED were the positive controls for HIV–1 protease. Similarly, NBV and cyclophellitol were the controls used for β–glucosidase and NOJ, N–methyl calystegine B2 for α–galactosidase. (Fig. 5) Ligands TGSB68 and TGSB482 had the energy value of –6.49 kcal/mol comparable to that of the average reference value of the positive control, and thus, the potent candidate as identified by molecular docking to HIV-1 protease. (Fig. 6a) The control compounds used for α–mannosidase were mannostatin and kifunensine, which bind with mean binding energy of -9.11 and -5.56. In the case of α–mannosidase, the same compounds TGSB68 and TGSB482 were selected due to comparable energy and a good cluster size with that of positive control. (Fig. 6b) For β– glucosidase, ligands TGSC108 and TGSC236, which had comparable values to that of positive control was identified as the Figure 5 Figure 6 potent candidate. (Fig. 6c) In the case of α–galactosidase, again the ligands TGSB68 and TGSB482 were selected based on binding energies. (Fig. 6d) In conclusion, the concept analogy (deoxy nature, planarity, thiosugar framework, lactone moiety) for the design of inhibitors indeed worked positively. The results are really encouraging. An in vivo study of the synthesized novel thiosugar probes will certainly provide a potent inhibitor. Section D entitled “Research methodology” provides experimental procedures adopted with details of synthesis. Section E entitled “Bibliography” provides the references cited in this work.

Dans un contexte où les iminosucres multivalents représentent, en tant qu’inhibiteurs puissants de glycosidases, des structures privilégiées pour le développement de nouveaux agents thérapeutiques, nous nous sommes intéressés à ce type de composés pour le traitement de deux maladies génétiques rares. Le premier axe de recherche a consisté à synthétiser des iminosucres di- à tétravalents en série 1-désoxymannojirimycine dans le but d’inhiber l’α1,2-mannosidase I du réticulum endoplasmique qui est impliquée dans la destruction de la protéine delF508-CFTR chez les malades atteints de la mucoviscidose. Un effet multivalent fort sur la correction de cette protéine mutée a alors été mis en évidence avec un composé trivalent basé sur le pentaérythritol. Efficace à des concentrations submicromolaires, ce dernier s’est montré 140 fois plus efficace que le modèle monovalent correspondant. Le second axe de recherche a consisté à identifier de nouveaux chaperons pharmacologiques de la β-glucocérébrosidase, l’enzyme lysosomale impliquée dans la maladie de Gaucher. Pour cela, nous avons préparé une série d’iminosucres hétérodivalents conçus pour cibler simultanément le site actif et un site secondaire de cette enzyme. Même si cet objectif n’a pas encore été atteint, nous avons malgré tout mis en évidence des chaperons monovalents capables de quasiment quadrupler l’activité de la β-glucocérébrosidase portant la mutation G202R. En marge de ces deux axes principaux, une sonde mécanistique basée sur un C-glycoside multivalent a également été développée dans le but de préciser les mécanismes à l’origine des effets multivalents puissants observés pour l’inhibition des glycosidases. / Because multivalent iminosugars represent, as potent glycosidase inhibitors, privileged structures for the design of novel drugs, we took a particular interest in this class of compounds for the treatment of two rare genetic diseases. The first research topic was dedicated to the synthesis of di- to tetravalent iminosugars in the 1-deoxymannojirimycin series in order to inhibit the endoplasmic reticulum α1,2-mannosidase I involved in the destruction of delF508-CFTR, the mutant protein responsible of cystic fibrosis. A strong multivalent effect for restoring its activity in cells was reported with a trivalent analogue based on pentaerythritol. This submicromolar corrector was found to be 140-fold more potent than the corresponding monovalent model. The second research topic focused on the identification of novel pharmacological chaperones of the β-glucocerebrosidase, the lysosomal enzyme involved in Gaucher’s disease. For this purpose, we developed a series of heterodivalent iminosugars designed to both bind to the active site and a secondary site of the enzyme. This goal could not be reached yet, nevertheless we identified monovalent chaperones which were able to fourfold increase β-glucocerebrosidase activity in G202R cell lines. Next to these main research topics, a mechanistic probe based on a multivalent C-glycoside was also developed to investigate the multivalent effect of iminosugar clusters in glycosidase inhibition.