Innovative chemical and chemoenzymatic synthesis of antigenic oligosaccharides towards vaccination against bacillary dysentery

Reflet d'un enjeu sanitaire important, les bactéries entéroinvasives Gram négatif de l'espèce Shigella flexneri représentent des cibles prioritaires dans le développement d'un vaccin diarrhéique. Cibles privilégiées de la protection contre la réinfection, les polysaccharides de surface - antigènes-O (Ags-O) - d'une grande majorité de S. flexneri dérivent d'un même squelette. Celui-ci est défini par une unité répétitive constituée de trois résidus L-rhamnose et un résidu N-acétyl-D-glucosamine (ABCD), liés en 1,2-trans. Les multiples patterns d'O-acétylation et/ou alpha-D-glucosylation régiospécifique de ce tétrasaccharide illustrent la diversité sérotypique. La synthèse de glycobriques modulables, aisées à combiner en un ensemble d'oligosaccharides biologiquement actifs représentatifs des Ags-O d'intérêt, est le fondement d'une stratégie visant le développement d'un vaccin conjugué dérivé de sucres de synthèse à large couverture contre Shigella. A cette fin, ces travaux explorent un concept original portant sur l'implémentation d'une voie de synthèse divergente pour accéder à un ensemble de glycobriques pré-sélectionnées. Cette stratégie met en oeuvre une étape d'alpha-D-glucosylation enzymatique, stéréo- et régiospécifique, de deux accepteurs de type ABCD, les tétrasaccharides ABC'D' et D'ABC'. Protégés a minima, ces accepteurs ont été conçus pour être (i) compatibles avec l'activité de glucanesucrases de la famille 70 des Glycosides Hydrolases, (ii) aisément accessibles et (iii) oligomérisables à leurs deux extrémités. Diverses stratégies de synthèse convergente exploitant les propriétés d'orthogonalité fine entre groupes protecteurs et la combinaison de deux blocs ABC et D, ont été évaluées. Les synthèses les plus prometteuses ont été optimisées et soumises à une montée en échelle visant à produire les cibles ABC'D' et/ou D'ABC'. La possible conversion de l'accepteur ABC'D' en différents produits d'alpha-D-glucosylation via l'action de glucanesucrases a été démontrée par nos collaborateurs du TBI (anciennement LISBP, Toulouse). Les données structurales ont révélé que certains pentasaccharides présentent les motifs caractéristiques d'Ags-O de S. flexneri. Nos résultats illustrent la synergie possible entre synthèse chimique et synthèse enzymatique dans le contexte Shigella, offrant de nouvelles options quant à l'obtention d'oligosaccharides de S. flexneri à visée vaccinale.Gram-negative bacteria from the Shigella flexneri species are a leading cause of diarrheal diseases in children under five in developing countries and prime targets for vaccine development. Most S. flexneri serotypes of concern exhibit closely related surface polysaccharides - O-antigens (O-Ags) - that are a main target of protection against reinfection. Their O-Ag backbones have in common a unique repeating unit made of three L-rhamnose and a N-acetyl-D-glucosamine residues (ABCD), 1,2-trans linked to one another. Structural diversity reflecting serotype specificity comprises site-selective O-acetylation and alpha-D-glucosylation. The synthesis of fine-tuned glycobricks, easy-to-assemble into biologically active oligosaccharides mimicking the O-Ag from selected serotypes, is the keystone of ongoing development to a broad coverage synthetic carbohydrate-based Shigella vaccine. Toward this aim, this work explores an original concept consisting in the regio- and stereospecific enzymatic alpha-D-glucosylation of two lightly protected ABCD-like tetrasaccharide acceptors, ABC'D' and D'ABC'. Suitable tetrasaccharides, masked at three positions, were designed to fulfill: (i) compatibility with the binding site topology of branching sucrases from the Glycoside Hydrolase family 70, (ii) easy access, and (iii) possible chemical elongation at either end post alpha-D-glucosylation. Highly convergent chemical strategies to the selected non-natural ABC'D' and D'ABC' tetrasaccharides were explored stemming from the combination of two versatile ABC and D building blocks and fine-tuning of orthogonal protecting groups. The most promising syntheses have been optimized and scaled up to achieve multigram amounts of the two acceptors of enzymatic glucosylation. The feasibility of the glucansucrase-mediated conversion of ABC'D' into several alpha-D-glucosylated products was demonstrated by our collaborators at TBI (previously LISBP, Toulouse). Structural characterization revealed that some of the isolated pentasaccharides featured the essential O-Ag type-specificity. Besides illustrating the possible synergy between chemical and enzymatic synthesis in the context of Shigella, our results broaden opportunities for the synthesis of S. flexneri oligosaccharides of interest for use in vaccine elaboration

Development of carbon monoxide-releasing molecules conjugated to polysaccharides (glyco-CORMs) for delivering CO during obesity

Metal carbonyls have been developed as carbon monoxide-releasing molecules (CO-RMs) to deliver CO for therapeutic purposes. The manganese-based CORM-401 has been recently reported to exert beneficial effects in obese animals by reducing body weight gain, improving glucose metabolism and reprogramming adipose tissue towards a healthy phenotype. Here, we report on the synthesis and characterization of glyco-CORMs, obtained by grafting manganese carbonyls on dextrans (70 and 40 kDa), based on the fact that polysaccharides facilitate the targeting of drugs to adipose tissue. We found that glyco-CORMs efficiently deliver CO to cells in vitro with higher CO accumulation in adipocytes compared to other cell types. Oral administration of two selected glyco-CORMs (5b and 6b) resulted in CO accumulation in various organs, including adipose tissue. In addition, glyco-CORM 6b administered for eight weeks elicited anti-obesity and positive metabolic effects in mice fed a high fat diet. Our study highlights the feasibility of creating carriers with multiple functionalized CO-RMs

Computer-aided engineering of a branching sucrase for the glucodiversification of a tetrasaccharide precursor of S. flexneri antigenic oligosaccharides

International audienceEnzyme engineering approaches have allowed to extend the collection of enzymatic tools available for synthetic purposes. However, controlling the regioselectivity of the reaction remains challenging, in particular when dealing with carbohydrates bearing numerous reactive hydroxyl groups as substrates. Here, we used a computer-aided design framework to engineer the active site of a sucrose-active α-transglucosylase for the 1,2-cis-glucosylation of a lightly protected chemically synthesized tetrasaccharide, a common precursor for the synthesis of serotype-specific S. flexneri O-antigen fragments. By targeting 27 amino acid positions of the acceptor binding subsites of a GH70 branching sucrase, we used a RosettaDesign-based approach to propose 49 mutants containing up to 15 mutations scattered over the active site. Upon experimental evaluation, these mutants were found to produce up to six distinct pentasaccharides, whereas only two were synthesized by the parental enzyme. Interestingly, we showed that by introducing specific mutations in the active site of a same enzyme scaffold, it is possible to control the regiospecificity of the 1,2-cis glucosylation of the tetrasaccharide acceptor and produce a unique diversity of pentasaccharide bricks. This work offers novel opportunities for the development of highly convergent chemo-enzymatic routes toward S. flexneri haptens

Redirecting substrate regioselectivity using engineered ΔN123-GBD-CD2 branching sucrases for the production of pentasaccharide repeating units of S. flexneri 3a, 4a and 4b haptens

International audienceThe (chemo-)enzymatic synthesis of oligosaccharides has been hampered by the lack of appropriate enzymatic tools with requisite regio-and stereo-specificities. Engineering of carbohydrate-active enzymes, in particular targeting the enzyme active site, has notably led to catalysts with altered regioselectivity of the glycosylation reaction thereby enabling to extend the repertoire of enzymes for carbohydrate synthesis. Using a collection of 22 mutants of ΔN 123-GBD-CD2 branching sucrase, an enzyme from the Glycoside Hydrolase family 70, containing between one and three mutations in the active site, and a lightly protected chemically synthesized tetrasaccharide as an acceptor substrate, we showed that altered glycosylation product specificities could be achieved compared to the parental enzyme. Six mutants were selected for further characterization as they produce higher amounts of two favored pentasaccharides compared to the parental enzyme and/or new products. The produced pentasaccharides were shown to be of high interest as they are precursors of representative haptens of Shigella flexneri serotypes 3a, 4a and 4b. Furthermore, their synthesis was shown to be controlled by the mutations introduced in the active site, driving the glucosylation toward one extremity or the other of the tetrasaccharide acceptor. To identify the molecular determinants involved in the change of ΔN 123-GBD-CD2 regioselectivity, extensive molecular dynamics simulations were carried out in combination with in-depth analyses of amino acid residue networks. Our findings help to understand the interrelationships between the enzyme structure, conformational flexibility and activity. They also provide new insight to further engineer this class of enzymes for the synthesis of carbohydrate components of bacterial haptens. Carbohydrate-active enzymes catalyze a wide range of chemical reactions. They have emerged as a practical alternative to chemical catalysts, avoiding multiple steps of protection and deprotection often required in chemical synthesis to control the reactivity of the sugar hydroxyl groups and regio-and stereo-selectivity of the reaction. Some of them are rather versatile biocatalysts often displaying naturally a relaxed substrate specificity. This promiscuity can be further exacerbated by enzyme engineering to either broaden or narrow down the range of recognized substrates and/or control the reaction selectivity 1. In particular, mutagenesis targeting the enzym

A convergent chemoenzymatic strategy to deliver a diversity of Shigella flexneri serotype-specific O-antigen segments from a unique lightly protected tetrasaccharide core

International audienceProgress in glycoscience is strongly dependent on the availability of broadly diverse tailored-made, well-defined and often complex oligosaccharides. Herein, going beyond natural resources and aiming to circumvent chemical boundaries in glycochemistry, we tackle the development of an in vitro chemoenzymatic strategy holding great potential to answer the need for molecular diversity characterizing microbial cell-surface carbohydrates. The concept is exemplified in the context of Shigella flexneri, a major cause of diarrheal disease. Aiming at a broad serotype coverage S. flexneri glycoconjugate vaccine, a non-natural lightly protected tetrasaccharide was designed for compatibility with (i) serotype-specific glucosylations and O-acetylations defining S. flexneri O-antigens, (ii) recognition by suitable α-transglucosylases, and (iii) programmed oligomerization post enzymatic -D-glucosylation. The tetrasaccharide core was chemically synthesized from two crystalline monosaccharide precursors. Six α-transglucosylases found in the Glycoside Hydrolase family 70 were shown to transfer glucosyl residues on the non-natural acceptor. The successful proof-of-concept is achieved for a pentasaccharide featuring the glucosylation pattern from the S. flexneri type IV O-antigen. It demonstrates the potential of appropriately planned chemo-enzymatic pathways involving non-natural acceptors and low-cost donor/transglucosylase systems to achieve the demanding regioselective -D-glucosylation of large substrates, paving the way to microbial oligosaccharides of vaccinal interest

Identification of a mechanism promoting mitochondrial sterol accumulation during myocardial ischemia–reperfusion: role of TSPO and STAR

International audienceHypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4’-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4’-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4’-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury