Conception de ligands à charpente saccharidique pour le ciblage multivalent et la vectorisation

Nous avons conçu et développé une méthode de synthèse de ligands multivalents modulables contenant une charpente saccharidique dans le but d étudier ou d inhiber les interactions impliquées dans différentes pathologies. Dans un premier temps, nous avons greffé des galactosides dont la longueur de la chaîne est variable sur cette charpente. Cela dans le but d étudier l influence de la longueur des bras espaceurs dans le processus d interactions multivalentes envers une lectine modèle. Ces composés seront testés par microscopie de force atomique. Dans un deuxième temps, nous avons greffé des mannosides d heptyle sur cette charpente dans le but d inhiber l adhésion de FimH, qui est une lectine importante dans le processus de fixation de la bactérie uropathologique E. coli sur les cellules du conduit urinaire. L affinité de nos composés a été évaluée par résonnance plasmonique de surface et par calorimétrie par titration isotherme envers la protéine, et également par inhibition d hémagglutinine sur des souches bactériennes. Nous avons montré que nos composés sont des inhibiteurs nanomolaires. Dans un troisième temps, nous avons fonctionnalisé sélectivement un peptide cyclique potentiellement antiangiogénique inhibant le récepteur 2 du VEGF. Différents agents chélatants utilisés en imagerie médicale ont été couplés à ce peptide par cycloaddition 1,3 dipolaire alcyne/azoture catalysé par le cuivre.We have been working on the design and synthesis of new multivalent ligands based on carbohydrate scaffolds as probes to investigate or inhibit interactions involved in different pathologies. First, galactoside ligands with different linker length were grafted on these scaffolds in order to study the influence of the linker length influence on the binding affinity toward a lectin. These compounds will be tested by Atomic Force Microscopy. Second, heptylmannoside ligands were tethered on these scaffolds in order to inhibit the FimH adhesion, an important lectin for the binding of uropathogenic E. coli to the urinary tract. The binding affinity of these compounds to the FimH and the bacteria were evaluated by Surface Plasmon Resonance, Haemagglutination Inhibition Assay and Isothermal Titration Calorimetry. Our compounds have shown to be nanomolar inhibitors. Third, a cyclic peptide that is a potent antiangiogenic compound by inhibiting VEGFr-2 receptor was selectively functionalized. Specific chelating agents used in medical imaging techniques, such as Magnetic Resonance Imaging or Single Photon Emission Computed Tomography were grafted onto the cyclic peptide by Copper Alkyne Azide Cycloaddition.AMIENS-BU Sciences (800212103) / SudocSudocFranceF

The impact of heteromultivalency in lectin recognition and glycosidase inhibition: an integrated mechanistic study

The vision of multivalency as a strategy limited to achieve affinity enhancements between a protein receptor and its putative sugar ligand (glycotope) has proven too simplistic. On the one hand, binding of a glycotope in a dense glycocalix-like construct to a lectin partner has been shown to be sensitive to the presence of a third sugar entity (heterocluster effect). On the other hand, several carbohydrate processing enzymes (glycosidases and glycosyltransferases) have been found to be also responsive to multivalent presentations of binding partners (multivalent enzyme inhibition), a phenomenon first discovered for iminosugar-type inhibitory species (inhitopes) and recently demonstrated for multivalent carbohydrate constructs. By assessing a series of homo- and heteroclusters combining α-d-glucopyranosyl-related glycotopes and inhitopes, it was shown that multivalency and heteromultivalency govern both kinds of events, allowing for activation, deactivation or enhancement of specific recognition phenomena towards a spectrum of lectin and glycosidase partners in a multimodal manner. This unified scenario originates from the ability of (hetero)multivalent architectures to trigger glycosidase binding modes that are reminiscent of those harnessed by lectins, which should be considered when profiling the biological activity of multivalent architectures.This study was supported by the Spanish Ministerio de Economía y Competitividad (contract numbers SAF2016‐76083‐R and CTQ2015‐64425‐C2‐1‐R), the Junta de Andalucía (contract number FQM2012‐1467) and the European Regional Development Funds (FEDER and FSE)

Heptyl α- D -Mannosides Grafted on a β-Cyclodextrin Core To Interfere with Escherichia coli Adhesion: An In Vivo Multivalent Effect

n‐Heptyl α‐D‐mannoside (HM) has previously been identified as a nanomolar FimH antagonist able to prevent Escherichia coli adhesion. We have designed mono‐ and heptavalent glycoconjugates in which HM is tethered to β‐cyclodextrin (β‐CD) through short and long spacers. One‐pot click or co‐clicking procedures were developed to directly obtain the glycoconjugates from unprotected HM and β‐CD precursors. These FimH antagonists were examined biophysically and in vivo. Reverse titrations by isothermal calorimetry led to trapping of the short‐tethered heptavalent β‐CD in a complex with three FimH lectins. Combined dynamic light scattering and small‐angle X‐ray solution scattering data allowed the construction of a model of the FimH trimer. The heptavalent β‐CDs were shown to capture and aggregate living bacteria in solution and are therefore also able to aggregate FimH when attached to different bacteria pili. The first in vivo evaluation of multivalent FimH inhibitors has been performed. The heptavalent β‐CDs proved to be much more effective anti‐adhesive agents than monovalent references with doses of around 2 μg instilled in the mouse bladder leading to a significantly decreased E. coli load. Intravenously injected radiolabeled glycoconjugates can rapidly reach the mouse bladder and >2 μg concentrations can easily be retained over 24 h to prevent fluxing bacteria from rebinding

Probing the Nature of the Cluster Effect Observed with Synthetic Multivalent Galactosides and Peanut Agglutinin Lectin

We designed a set of multigalactosides with valencies ranging from one to seven and different spacer-arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme-linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer-arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross-linkers in a ``sand-wich assay''. Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates be-tween heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency-controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites