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Les publications de l’UMR

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Tobaldi, E., Dovgan, I., Mosser, M., Becht, JM., and Wagner, A.

Structural investigation of cyclo-dioxo maleimide cross-linkers for acid and serum stability, Org. Biol. Chem., 2017, 15, 9305-9310.

The biochemical characteristics of hetero-bifunctional cross-linkers used in bioconjugates are of essential importance to the desired features of the final adduct (i.e. antibody–drug conjugates). These include stability in biological media, chemical and biological reactivities, cleavability under defined conditions, and solubility. In our previous work, we introduced a new amino-to-thiol linker, maleimidomethyl dioxane (MD), as an alternative to classical maleimide conjugation, with increased hydrophilicity and serum stability due to succinimidyl ring-opening. In this work, we investigate the generality of linkers containing a dioxo-ring with regard to their ability to self-hydrolyze and their surprising stability at a low pH. We synthesized four FRET probes which allowed us to address the stability of the dioxo-ring and to study the maleimide ring-opening and the thiol-exchange processes by means of detecting and measuring the generation of fluorescence. It was found that the ring expansion (from a 5- to a 6-membered ring) improved the stability of the probes in aqueous media, and the increase of the chain length between the dioxo-ring and the succinimide ring (from methylene to ethylene) decreased the rate of succinimidyl ring-opening.

Exporter la référence : Org. Biomol. Chem.
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Dovgan, I., Erb, S., Hessman, S., Ursuegui, S., Michel, C., Muller, C., Chaubet, G., Cianférani, S., Wagner, A.

Arginine-selective bioconjugation with 4-azidophenyl glyoxal: application to the single and dual functionalisation of native antibody, Org. Biomol. Chem., 2018, 16, 1305-1311.

Here, we introduce 4-azidophenyl glyoxal (APG) as an efficient plug-and-play reagent for the selective functionalisation of arginine residues in native antibodies. The selective reaction between APG and arginines’ guanidine groups allowed a facile introduction of azide groups on the monoclonal antibody trastuzumab (plug stage). These pre-functionalised antibody–azide conjugates were then derivatised during the “play stage” via a biorthogonal cycloaddition reaction with different strained alkynes. This afforded antibody-fluorophore and antibody–oligonucleotide conjugates, all showing preserved antigen selectivity and high stability in human plasma. Due to a lower content of arginines compared to lysines in native antibodies, this approach is thus attractive for the preparation of more homogeneous conjugates. This method proved to be orthogonal to classical lysine-based conjugation and allowed straightforward generation of dual-payload antibody.

Exporter la référence : Organic & Biomolecular Chemistry
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Koniev, O. , Dovgan, I. , Renoux, B., Ehkirch, A. , Eberova, J. , Cianférani, S., Kolodych, S., Papot, S., Wagner, A.

Reduction–rebridging strategy for the preparation of ADPN-based antibody–drug conjugates, Med. Chem. Commun., 2018, 9, 827-830.

The reduction–rebridging strategy is a powerful method for the preparation of stable and homogeneous antibody–drug conjugates (ADCs). In this communication, we describe the development of the arylene-dipropiolonitrile (ADPN) functional group for the rebridging of reduced disulphide bonds and its application in the preparation of potent and selective ADCs.

Exporter la référence : MedChemComm
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Schaeffer, E., Flacher, V. , Neuberg, P., Hoste, A., Brulefert, A., Fauny, JD., Wagner A., Mueller, CG.

Inhibition of dengue virus infection by mannoside glycolipid conjugates, Antiviral. Res., 2018, 154, 116-123.

Dengue virus (DENV), a mosquito-borne flavivirus, causes severe and potentially fatal symptoms in millions of infected individuals each year. Although dengue fever represents a major global public health problem, the vaccines or antiviral drugs proposed so far have not shown sufficient efficacy and safety, calling for new antiviral developments. Here we have shown that a mannoside glycolipid conjugate (MGC) bearing a trimannose head with a saturated lipid chain inhibited DENV productive infection. It showed remarkable cell promiscuity, being active in human skin dendritic cells, hepatoma cell lines and Vero cells, and was active against all four DENV serotypes, with an IC50 in the low micromolar range. Time-of-addition experiments and structure-activity analyses revealed the importance of the lipid chain to interfere with an early viral infection step. This, together with a correlation between antiviral activity and membrane polarization by the lipid moiety indicated that the inhibitor functions by blocking viral envelope fusion with the endosome membrane. These finding establish MGCs as a novel class of antivirals against the DENV.

Exporter la référence : Antiviral Research
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Chaubet, G., Thoreau, F., Wagner, A.

Recent, non-classical, approaches to antibody lysine modification, Drug Discov. Today Technol., 2018, DOI: 10.1016/j.antiviral.2018.04.005.

This review will discuss recent development in the bioconjugation of lysine residues on antibodies. As several chemoselective reagents have already been developed for modifying amine groups, recent strategies now tend to aim at being site-specific. Four general methods have been listed: kinetically controlled, template-directed or enzymatic strategies as well as the use of chemically programmed antibodies.

Exporter la référence : Drug Discov. Today Technol.
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Nothisen, M., Perche-Létuvée, P., Behr, JP., Remy, JS., Kotera, M.

Cationic oligospermine-oligonucleotide conjugates provide carrier-free splice switching in monolayer cells and in spheroids,

We report the evaluation of 18-mer 2’-O-methyl-modified ribose oligonucleotides with a full-length phosphorothioate backbone chemically conjugated at the 5’-end to the oligospermine units [Sn-: n = 5, 15, 20, 25, 30 (number of spermine units)] as splice switching oligonucleotides (SSOs). These conjugates contain, in their structure, covalently linked oligocation moieties, making them capable of penetrating cells without transfection vector. In cell culture, we observed efficient cytoplasmic and nuclear delivery of fluorescein labelled S20-SSO by fluorescent microscopy. The SSO conjugates containing more than 15 spermine units induced significant carrier-free exon-skipping at nanomolar concentration in the absence and in the presence of serum. With an increasing number of spermine units, the conjugates became slightly toxic but more active. Advantages of these molecules were particularly demonstrated in 3D cell culture (MCTS: multicellular tumor spheroids) that mimics living tissues. While vector-complexed SSOs displayed a drastically reduced splice switching in MCTS compared to the assay in monolayer culture, an efficient exon-skipping without significant toxicity was observed with oligospermine grafted SSOs (S15- and S20-SSOs) transfected without vector. It was shown, by flow cytometry and confocal microscopy, that the fluorescein-labelled S20-SSO was freely diffusing and penetrating the innermost cells of MCTS while the vector-complexed SSO penetrated only the cells of the spheroid's outer layer.

Exporter la référence : Molecular Therapy: Nucleic Acids
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Muñoz, A., Sigwalt, D., Illescas, B. M., Luczkowiak, J., Rodriguez, L., Nierengarten, I., Holler, M., Remy, J.S., Buffet, K., Vincent, S. P., Rojo, J., Delgado, R., Nierengarten, J.F., Martin, N.

Synthesis of giant globular multivalent glycofullerenes as potent inhibitors in a model of Ebola virus infection, Nature Chemistry, 2016, 8, 50-57.

The use of multivalent carbohydrate compounds to block cell-surface lectin receptors is a promising strategy to inhibit the entry of pathogens into cells and could lead to the discovery of novel antiviral agents. One of the main problems with this approach, however, is that it is difficult to make compounds of an adequate size and multivalency to mimic natural systems such as viruses. Hexakis adducts of [60]fullerene are useful building blocks in this regard because they maintain a globular shape at the same time as allowing control over the size and multivalency. Here we report water-soluble tridecafullerenes decorated with 120 peripheral carbohydrate subunits, so-called ‘superballs’, that can be synthesized efficiently from hexakis adducts of [60]fullerene in one step by using copper-catalysed azide–alkyne cycloaddition click chemistry. Infection assays show that these superballs are potent inhibitors of cell infection by an artificial Ebola virus with half-maximum inhibitory concentrations in the subnanomolar range.

Exporter la référence : Nature Chemistry
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Jacques, SA., Leriche, G., Mosser, M., Nothisen, M., Muller, CD., Remy, JS., Wagner, A.

From solution to in-cell study of the chemical reactivity of acid sensitive functional groups: a rational approach towards improved cleavable linkers for biospecific endosomal release, Organic & biomolecular chemistry, 2016, 14, 4794-4803.

pH-sensitive linkers designed to undergo selective hydrolysis at acidic pH compared to physiological pH can be used for selective release of therapeutics at their site of action. In this paper, the hydrolytic cleavage of a wide variety of molecular structures that have been reported for their use in pH-sensitive delivery systems was examined . A wide variety of hydrolytic stability profiles was found among the panel of tested chemical functionalities. Even within a structural family, a slight modification of the substitution pattern has an unsuspected outcome on the hydrolysis stability. This work lead us to establish a first classification of these groups based on their reactivities at pH 5.5 and their relative hydrolysis at pH 5.5 vs. pH 7.4. From this classification, four representative chemical functions belonging eand compare lysosomal cleavage in living cells..and revealed that only the most reactive functions underwent significant lysosomal cleavage, according to flow cytometry measurements. These last results question the acid-based mechanism of action of known drug release systems and advocate for the importance of in-depth structure- reactivity study, using tailored methodology, for the rational design and development of bio-responsive linkers.

Exporter la référence : Organic & Biomolecular Chemistry
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Nothisen, M., Bagilet, J., Behr, JP., Remy, JS., Kotera, M.

Structure Tuning of Cationic Oligospermine–siRNA Conjugates for Carrier-Free Gene Silencing, Molecular Pharmaceutics, 2016, 13, 2718-2728.

Oligospermine−siRNA conjugates are able to induce efficient luciferase gene silencing upon carrier-free transfection. These conjugates are readily accessible by a versatile automated chemistry that we developed using a DMT-spermine phosphor- amidite reagent. In this article, we used this chemistry to study a wide range of structural modifications of the oligospermine−siRNA conjugates, i.e., variation of conjugate positions and introduction of chemical modifications to increase nuclease resistance. At first we examined gene silencing activity of a series of siRNA−tris(spermine) conjugates with and without chemical modifications in standard carrier assisted conditions. The three spermine units attached at one of the two ends of the sense strand or at the 3′-end of the antisense strand are compatible with gene silencing activity whereas attachment of spermine units at the 5′-end of the antisense strand abolished the activity. 2′-O-Methylated nucleotides introduced in the sense strand are compatible while not in the antisense strand. Thiophosphate links could be used without activity loss at the 3′-end of both strands and at the 5′-end of the sense strand to conjugate oligospermine. Consequently a series of oligospermine−siRNA conjugates containing 15 to 45 spermines units in various configurations were chosen, prepared, and examined in carrier-free conditions. Attachment of 30 spermine units singly at the 5′-end of the sense strand provides the most potent carrier-free siRNA. Longevity of luciferase gene silencing was studied using oligospermine−siRNA conjugates. Five day long efficiency with more than 80% gene expression knockdown was observed upon transfection without vector. Oligospermine−siRNA conjugates targeting cell-constitutive natural lamin A/C gene were prepared. Efficient gene silencing was observed upon carrier-free transfection of siRNA conjugates containing 20 or 30 spermine residues grafted at the 5′-end of the sense strand.

Exporter la référence : Molecular Pharmaceutics
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Ripoll, M., Neuberg, P., Kichler, A., Tounsi, N., Wagner, A., and Remy, JS.

Ph-responsive nanometric polydiacetylenic micelles allow for efficient intracellular siRNA delivery, ACS applied materials & interfaces, 2016, 45, 30665-30670.

A novel generation of pH-responsive photo- polymerized diacetylenic amphiphile (PDA) micelles with a diameter of 10 nm was designed and optimized for the intracellular delivery of siRNAs. Dialysis and photopolymeriza- tion of the micelles allowed a strong reduction of the cytotoxicity of the nanovector, while the hydrophilic histidine headgroup permitted enhancing the siRNA delivery potential by improving the endosomal escape via imidazole protonation. These PDA-micellar systems were fully characterized by DLS, TEM, and DOSY-NMR experiments. The resulting bioactive complexes of PDA-micelles with siRNA were shown to have an optimal size below 100 nm.

Exporter la référence : ACS Applied Materials & Interfaces
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