Catalysis, ORganometallic chemistry And synthesIs of Ligands
Glycochemistry & Bioconjugates Group
Glycochemistry & Bioconjugate Group ” (2017-2018) from left to right: Christophe Dussouy Post doc, Aurélien Leray PhD, Coralie Assailly PhD, Yoan Brissonnet Post doc, Elodie Langaraday Post doc, Roxane Peumery, Karine Julienne MCU, Valérie Fargeas MCU, Mathieu Mevel CDD, David Deniaud Prof, Sébastien Gouin DR CNRS.
Principal Investigator : Sébastien Gouin
Research activities :
Our research activities are focused on the development of therapeutically relevant glycoclusters (multivalent lectin and glycosidase modulators), glyco-coated virus for gene therapy and radio-conjugates.
Scientific highlights (2018-2019)
glyco-coated virus : Mathieu MEVEL HdR defense (18/03/2019)
Sugar probes: In the framework of the ANR project HICARE, we showed that polymeric thiosialosides inhibit with high synergy sialidases from T.cruzi and S. pneumoniae . These results extend the multivalent concept to the inhibition of bacterial and pathogenic sialidases for which transition-state inhibitors fail to reach the submicromolar level. Hot Paper, Chem. Eur. J. 2019 , 25 , 2358. https://onlinelibrary.wiley.com/doi/10.1002/chem.201805790
Bioconjugation : In collaboration with the group of Mohammed Boujtita (CEISAM) we developed a traceless electrochemical method for the bioconjugation of tyrosine residues from proteins. J. Am. Chem. Soc. 2018 , 140 , 17120. https://pubs.acs.org/doi/10.1021/jacs.8b09372
Sugar therapeutics : We developed and patented hexavalent antagonists of the FleA adhesin from the opportunistic mold Aspergillus fumigatus causing fatal lungs infections.
Collaboration: P. Le Pape (IICIMED); A. Varrot (CERMAV) Funding from SATT Ouest valorisation; Patent EP 17156 368.7. Pub: Chem. Eur. J. 2018 , 24 , 19243. https://onlinelibrary.wiley.com/doi/10.1002/chem.201803602
Bacterial resistances to antibiotics is a global health problem getting worst. In this context, we are developing mono- and multivalent carbohydrate-based anti-adhesive compounds to block the attachment of virus, bacteria, and mycobacteria to host cells.
5 selected articles:
Multivalent fucosides with nanomolar affinity for the Aspergillus fumigatus lectin FleA prevent spore adhesion to pneumocytes
Victor Lehot, Yoan Brissonnet, Christophe Dussouy, Sami Brument, Aurore Cabanettes, Emilie Gillon, David Deniaud, Annabelle Varrot, Patrice Le Pape, and Sébastien G. Gouin*, Chem. Eur. J. 2018 , 24 , 19243-19249.
The FleA receptor in the opportunistic pathogen Aspergillus fumigatus was shown to be involved in spore (conidium) adherence to airway mucins and bronchoepithelial cells. We developed multivalent fucosides with various valencies and spacer arm lengths as conidium anti-adhesives. The hexa- and octavalent fucosides were nanomolar antagonists of FleA, strongly inhibiting conidium adherence to pneumocytes.
Physiochemical tuning of potent E. coli antiadhesives by microencapsulation and homologation ;
Dorta, D.A.; Chalopin, T.; Sivignon, A.; de Ruyck, J.; Dumych, T.I.; Bilyy, R.O.; Deniaud, D.; Barnich, N.; Bouckaert, J.; Gouin, S.G.* ChemMedChem, 2017 , 12 , 986-998 .
We developed a new generation of potent anti-adhesives of pathogenic E. coli strains promoting the gut inflammation in Crohn's disease. The neo-thiazolylmannosides were homologated by a methyl group and included in a γ-cyclodextrin to form a water soluble NeoTazMan@γCD complex with retained anti-adhesive effect against E.coli .
The anti-adhesive strategy in crohn's disease: orally active mannosides to decolonize pathogenic Escherichia coli from the gut.
Alvarez Dorta, D.; Sivignon, A.; Chalopin, T.; Dumych,T.; Pecina, A.; Roos, G.; Bilyy, R.; Deniaud, D.; de Ruyck, J.; Bouckaert, J.; Barnich, N.; Gouin S. G.* ChemBioChem, 2016 , 17, 1-18 .
|Ready for take off? Adherent-invasive E. coli (AIEC) were shown to promote the inflammation of the intestinal mucosa of patients with Crohn's disease (CD). In this work, we designed a small library of AIEC antiadhesives. A lead compound was shown to decolonize AIEC from the gut of a transgenic mouse model of CD after oral administration. The water-soluble and non-cytotoxic compounds developed may open new perspectives in the treatment of CD.|
Thiazolylaminomannosides as potent anti-adhesives of type 1 piliated Escherichia coli isolated from Crohn's disease patients.
Brument, S.; Sivignon, A.; Dumych, T. I.; Moreau, N.; Roos, G.; Guérardel, Y.; Chalopin, T.; Deniaud, D.; Bilyy,R. O. ; * Darfeuille-Michaud, A.; * Bouckaert, J.; * Gouin, S. G. * J. Med. Chem, 2013 , 56 , 5395-5406 .
|Adherent-invasive Escherichia coli (AIEC) have previously been shown to induce gut inflammation in patients with Crohn's disease (CD). We developed a set of mannosides to prevent AIEC attachment to the gut by blocking the FimH bacterial adhesin. The crystal structure of the FimH lectin domain in complex with a lead thiazolylaminomannoside highlighted the preferential position for pharmacomodulations. A small library of analogues showing nanomolar affinity for FimH was then developed. Notably, AIEC attachment to intestinal cells was efficiently prevented by the most active compound and at around 10000-fold and 100-fold lower concentrations than mannose and the potent FimH inhibitor heptylmannoside, respectively. An ex vivo assay performed on the colonic tissue of a transgenic mouse model of CD confirmed this antiadhesive potential. Given the key role of AIEC in the chronic intestinal inflammation of CD patients, these results suggest a potential antiadhesive treatment with the FimH inhibitors developed.|
Heptyl α-D-mannosides grafted on a β-cyclodextrin core to interfere with Escherichia coli adhesion – an in vivo multivalent effect.
Bouckaert, J. *; Zhaoli; Li.; Xavier, C.; Almant, M.; Caveliers; V.; Lahoutte, T.; Weeks, S.; Kovensky, J; Gouin, S.G. * Chem. Eur. J. 2013 , 24, 7847-7855.
|Let's stick together . One pot “click or co-clicking” procedures were developed to directly obtain mono- and heptavalent conjugates from unprotected alkynyl-armed heptyl mannoside (HM) and azido- ? -CD synthons. These anti-adhesive were shown to aggregate both the FimH adhesin and uropathogenic bacteria in solution. The first in vivo evaluation of synthetic multivalent FimH inhibitors was performed. The heptavalent ? -CD proved to be much more effective anti-adhesive agents than monovalent HM references, with doses of around 2 µg instilled in the mouse bladder leading to a significantly decreased E. coli load. Intravenously injected radiolabelled glycoconjugate can rapidly reach the mouse bladder and are retained over 24 h.|
Multivalent ligands are extensively used to inhibit carbohydrate-binding proteins (lectins). We have transferred this concept to carbohydrate-processing enzymes (glycosidases), by synthesizing and showing that multivalent iminosugars could inhibit or boost enzymatic activities.
5 selected articles:
Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases.
Brissonnet, Y. ; Assailly, C.; Saumonneau, A. ; Bouckaert, J.; Maillasson, M.; Petitot, C.; Roubinet, B.; Didak, B.; Landemarre, L.; Bridot, C.; Blossey, R.; Deniaud, D.; Yan, X.; Bernard, J.; Tellier, C.; Grandjean, C.; Daligault, F.; Gouin S.G. Hot Paper, Chem. Eur. J. 2019 , 25 , 2358-2365 .
High synergy: Sialidases (SA) are virulence factors expressed by pathogenic bacteria, viruses and parasites. Di- and polymeric thiosialosides were designed to inhibit V. cholerae , T. cruzi and S. pneumoniae sialidases. Each thiosialoside grafted on the polymer was shown to reduce SA catalytic activity with much higher efficiency compared to their monovalent analogues. This extend the multivalent concept to this class of enzymes.
Magnetic nanoparticules coated with thiomannosides or iminosugars to switch and recycle galactosidase activity ;
Alvarez-Dorta, D.; Brissonnet, Y.; Saumonneau, A.; Deniaud, D.; Bernard, J.; Tellier, C. ; Daligault, F. ;Gouin, S.G. * Chemistry Select , 2017 , 2 , 9552-9556.
Speed 2. Magnetic nanoparticles coated with thiomannosides (SMan@Fe3O4 ) and deoxynojirimycin iminosugars (DNJ@Fe3O4) switched a-galactosidase (aGal) activity from strong inhibition to activation profiles, respectively. The αGal-DNJ@Fe3O4complex could be magnetically recovered and conserve a higher catalytic velocity than αGal alone during the first four cycles.
Multivalency to potently inhibit and discriminate between hexosaminidase .
Alvarez-Dorta, D.; King, D.; Legigan, T.; Kato, A.; Désiré, J.; Deniaud, D.; Vocadlo, D.; Gouin,* S.G.; Blériot,* Y. Chem. Eur.J. 2017 , 23 , 9022-9025 .
|All crowded round : Multivalent acetamidoazepanes were designed to assess the effect of multivalency against biologically relevant β-hexosaminidases. Nanomolar multivalent inhibitors of HexAB, with improved enzyme selectivity, could be obtained by clustering a micromolar azepan inhibitor. These results extend the multivalent concept to an important class of glycosidases.|
Polymeric iminosugars to improve the activity of carbohydrate-processing enzymes.
Brissonnet,Y.; Tezé, D.; Fabre, E.; Deniaud, D.; Daligault, F.; Tellier, C.; Šesták, S.; Ladévèze , S.; Remaud-Simeon , M.; Potocki- Véronèse, G.; Gouin, S. G. * Bioconjugate chem. 2015 , 26 , 766-772 .
|Multivalent iminosugars have recently emerged as powerful tools to inhibit the activities of specific glycosidases. In this work, biocompatible dextrans were coated with iminosugars to form linear and ramified polymers with unprecedently high valencies (from 20 to 900) to probe the evolution of the multivalent inhibition as a function of ligand valency. This study led to the discovery that polyvalent iminosugars can also significantly enhance, and not only inhibit, the enzymatic activity of specific glycoside-hydrolase, as observed on two galactosidases, a fucosidase, and a bacterial mannoside phosphorylase for which an impressive 70-fold activation was even reached. The concept of glycosidase activation is largely unexplored, with a unique recent example of small-molecules activators of a bacterial O-GlcNAc hydrolase. The possibility of using these polymers as “artificial enzyme effectors” may therefore open up new perspectives in therapeutics and biocatalysis|
Topological effects and binding modes operating with multivalent iminosugar-based glycoclusters and glycosidases .
Brissonnet Y., Ortiz-Mellet C., Morandat S., Garcia Moreno I., Deniaud D., Matthews S. E., Vidal S., El Kirat K., Gouin. S. G. * J. Am. Chem. Soc. 2013 , 135 , 18427-18435.
Multivalent iminosugars have been recently explored for glycosidase inhibition. Affinity enhancements due to multivalency have been reported for specific targets, which are particularly appealing when a gain in enzyme selectivity is achieved but raise the question of the binding mode operating with this new class of inhibitors. Here we describe the development of a set of tetra- and octavalent iminosugar probes with specific topologies and an assessment of their binding affinities toward a panel of glycosidases including the Jack Bean α-mannosidase (JBαMan) and the biologically relevant class II α-mannosidases from Drosophila melanogaster belonging to glycohydrolase family 38, namely Golgi α-mannosidase ManIIb (GM) and lysosomal α-mannosidase LManII (LM). Very different inhibitory profiles were observed for compounds with identical valencies, indicating that the spatial distribution of the iminosugars is critical to fine-tune the enzymatic inhibitory activity. Compared to the monovalent reference, the best multivalent compound showed a dramatic 800-fold improvement in the inhibitory potency for JBαMan, which is outstanding for just a tetravalent ligand. The compound was also shown to increase both the inhibitory activity and the selectivity for GM over LM. This suggests that multivalency could be an alternative strategy in developing therapeutic GM inhibitors not affecting the lysosomal mannosidases. Dynamic light scattering experiments and atomic force microscopy performed with coincubated solutions of the compounds with JBaMan shed light on the multivalent binding mode. The multivalent compounds were shown to promote the formation of JBαMan aggregates with different sizes and shapes. The dimeric nature of the JBαMan allows such intermolecular cross-linking mechanisms to occur.
We develop new bioconjugation methods and radioconjugates for cancer imaging or therapy and metal decorporation. More particularly, we developed iron complexes of polyamines as cancer cells antiproliferative compounds, astatin 211 complexes for cancer radioimmunotherapy, and chelators of polonium 210 (antidote).
5 selected articles:
Electrochemically promoted tyrosine-click-chemistry for protein labelling
Dimitri Alvarez-Dorta, Christine Thobie-Gautier, Mikael Croyal, Mohammed Bouzelha, Mathieu Mével, David Deniaud, Mohammed Boujtita, and Sébastien G. Gouin. J. Am. Chem. Soc. 2018 , 140 , 17120. .
The development of new bioorthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazols. A low potential is applied in an electrochemical cell to activate PTAD anchors in situ and on demand, without affecting the electroactive amino-acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.
Investigation of a new “N2S2O2” chelating agent with high Po(IV) affinity
Younes A.; Montavon G.; Gouin S.G.; André-Joyaux E.; Peumery R.; Chalopin T.; Alliot C.; Mokili M.; Champion J.; Deniaud D. Chem. Comm ., 2017 , 53, 6492-6495.
A water-soluble “N2S2O2” complexing agent was designed for polonium(IV) decorporation. The bifunctional ligand showed outstanding Po(IV) complexing abilities, with a conditional stability constant three orders of magnitude higher than the reference ligand BAL .
Targeted radionuclide therapy with astatine-211: Oxidative dehalogenation of astatobenzoate conjugates
Teze D.; SergentuD-C.; Kalichuk V.; Barbet J.; Deniaud D .; Galland N.; Maurice R.; Montavon G. Scientific Reports , 2017 , 7, article number 2579.
|211At is a most promising radionuclide for targeted alpha therapy. However, its limited availability and poorly known basic chemistry hamper its use. Based on the analogy with iodine, labelling is performed via astatobenzoate conjugates, but in vivo deastatination occurs, particularly when the conjugates are internalized in cells. Actually, the chemical or biological mechanism responsible for deastatination is unknown. In this work, we show that the C-At “organometalloid” bond can be cleaved by oxidative dehalogenation induced by oxidants such as permanganates, peroxides or hydroxyl radicals. Quantum mechanical calculations demonstrate that astatobenzoates are more sensitive to oxidation than iodobenzoates, and the oxidative deastatination rate is estimated to be about 6x106 faster at 37 °C than the oxidative deiodination one. Therefore, we attribute the “internal” deastatination mechanism to oxidative dehalogenation in biological compartments, in particular lysosomes.|
Review Recent advances in cancer treatment by iron chelators
Corcé V.; Gouin S.G.; Renaud S.; Gaboriau F.; Deniaud D. Bioorg. Med. Chem. Lett . 2016 , 26, 251-256.
|The development of new therapeutic alternatives for cancers is a major public health priority. Among the more promising approaches, the iron depletion strategy based on metal chelation in the tumoral environment has been particularly studied in recent decades. After a short description of the importance of iron for cancer cell proliferation, we will review the different iron chelators developed as potential chemotherapeutics. Finally, the recent efforts to vectorize the chelating agents specifically in the microtumoral environment will be discussed in deta|
Synthesis and characterization of a stable copper(I) complex for radiopharmaceutical applications
Bodio E.; Boujtita M.; Julienne K.; Le Saec P.; Gouin S.G.; Hamon J.; Renault E.; Deniaud D. ChemPlusChem , 2014 , 79, 1284-1293.
N 4 Cu complexes : A series of ligands has been synthesized and their complexation with copper(II) precursors studied. The combination of several analytical techniques and density functional calculations shows that N4 copper(I) complexes are formed from a copper(II) salt (see figure). Preliminary tests of complexation show the ability of these ligands to chelate 64Cu in very diluted medium, which is of interest for developing new positron emission tomography imaging agents.
Synthesis and Biological Properties of Quilamines II, New Iron Chelators with Antiproliferative Activities
Corcé V.; Renaud S.; Cannie I.; Julienne K.; Gouin G.; Loréal O.; Gaboriau F.; Deniaud, D. Bioconjugate. Chem ., 2014 , 25 (2), 320-334.
To selectively target tumor cells expressing an overactive Polyamine Transport System (PTS), we designed, synthesized and evaluated the biological activity of a new generation of iron chelators, derived from the lead compound HQ1-44 , which we named Quilamines II. The structures of four new antiproliferative agents were developed. They differ in the size of the linker ( HQ0-44 and HQ2-44 ) or in the nature of the linker ( HQCO-44 and HQCS-44 ) between a hydroxyquinoline moiety (HQ) and a homospermidine (44) chain, the best polyamine vector . The Quilamines II were obtained after 6 to 9 steps by Michael addition, peptide linkage and reductive amination or by using the Willgerodt-Kindler reaction. The biological evaluation of these second-generation Quilamines showed that modifying the size of the linker increased the selectivity of these compounds for the PTS. In addition, measurement of the toxicity of Quilamines HQ0-44 and HQ2-44 highlighted their marked antiproliferative nature on several cancerous cell lines as well as a differential activity on non-transformed cells (fibroblasts). In contrast, Quilamines HQCO-44 and HQCS-44 presented low selectivity for the PTS, probably due to a loss of electrostatic interaction. We also demonstrated that the HCT 116 cell line, originating from a human colon adenocarcinoma, was the most responsive to the various Quilamines. As deduced from the calcein and HVA assays, the higher iron chelating capacity of HQ1-44 could explain its higher antiproliferative efficiency .
Our group also focuses on the development of novel, chemically modified AAV particles, with increased therapeutic effect, at the interface between vectorology and chemistry. AAVs are efficient therapeutic platforms for the treatment of genetic diseases. Yet, past and ongoing clinical trials have highlighted some limitations, including the need for high doses to achieve therapeutic benefit, and the off-target transduction of various tissues. Chemically modified AAVs should enable to greatly decrease off-target effects, hence maximizing transduction in tissues of interest and improving the therapeutic index in AAV-based gene therapy. This will be possible through covalent coupling of tissue-specific ligands on surface exposed residues of the AAV capsid. The main advantage of this chemistry oriented approach is to be able to functionalize AAV particles with various ligands which could not be genetically encoded, including polymers, sugars or lipids.
Recombinant AAV vectors are now becoming therapeutic products. However, several clinical trials using AAV have shown critical limitations: (i) high doses are usually required to achieve therapeutic effect; (ii) broad biodistribution to non-target tissues ; (iii) loss of efficacy in the presence of pre-existing neutralizing antibodies.
In an attempt to overcome these barriers, here we have used organic chemistry to improve the “specific activity” and the “therapeutic index” of rAAVs. As a proof of concept, a fluorescent probe was used to demonstrate the covalent coupling between an AAV2 and an organic molecule. For this purpose, the fluorophore molecule FITC, that contains an isothiocyanate chemical function (-NCS), was grafted on AAV2 particle by reacting with amino groups present at the surface of the capsid. Multiple analytical tools were used to show, unambiguously, a covalent coupling of the FITC molecule to the surface of the capsid by formation of a thiourea function and not an adsorption of this molecule. Notably, we also demonstrated that it is possible to modulate the number of FITC molecules grafted to the AAV particles according to the experimental conditions used in the chemical reaction (i.e. number of FITC Equivalents (Eq)/ AAV particles) (A). Using confocal microscopy it was possible to visualize FITC-AAV2 particles at the intracellular level upon transduction of HeLa cells (B). Interestingly, when AAV2 particles were labeled with high amounts of FITC (3e6 Eq/AAV particle), we noted an absence of recognition by the A20 antibody, suggesting that chemical modifications could decrease the sensitivity of Chem-AAV to preexisting neutralizing antibodies and/or impair the humoral response to the modified capsid for in vivo studies.
Based on these encouraging results, we decided to use our chemical modification technology to increase the specific targeting of AAV towards hepatocytes. To this end, N -acetylgalactosamine ligand (GalNAc) with a -NCS coupling function was synthesized since GalNAc recognizes the asialoglycoprotein receptor (ASPGR) present at the surface of hepatocytes. Dot and Western blot analysis showed higher amounts of GalNAc molecules covalently coupled on the surface of the AAV2 capsid by using increasing amounts of ligands (3e5 vs 3e6 Eq) (C). Importantly, GalNAc-AAV2 particles were more efficient in transducing mice primary hepatocytes than wild-type AAV2 and the efficacy was boosted by using increasing amounts of ligands (D). Altogether, our data shows the possibility to modulate at will the quantity and type of targeting ligands at the surface of the AAV particles and opens novel possibilities for gene transfer.