RESUMO
Following an iterative oxime ligation procedure, cyclopeptide (R) and lysine-based dendron (D) were combined in all possible arrangements and successively functionalized with α-fucose and ß-fucose to provide a new series of hexadecavalent glycosylated scaffolds (i.e., scaffolds RD16, RR16, DR16, and DD16). These compounds and smaller analogs (tetra- and hexavalent scaffolds R4 and R6) were used to evaluate the influence of the ligand valency and architecture, and of the anomer configuration in the binding to the αFuc-specific lectin LecB from Pseudomonas aeruginosa . Competitive enzyme-linked lectin assays (ELLA) revealed that only the RD16 architecture displaying αFuc (9A) reaches strong binding improvement (IC50 of 0.6 nM) over αMeFuc, and increases the α-selectivity of LecB. Dissociation constant of 28 nM was measured by isothermal titration micorcalorimetry (ITC) for 9A, which represents the highest affinity ligand ever reported for LecB. ITC and molecular modeling suggested that the high affinity observed might be due to an aggregative chelate binding involving four sugar head groups and two lectins. Interestingly, unprecedented binding effects were observed with ß-fucosylated conjugates, albeit being less active than the corresponding ligands of the αFuc series. In particular, the more flexible lysine-based dendritic structures (15B and 18B) showed a slight inhibitory enhancement in comparison with those having cyclopeptide core.
Assuntos
Dendrímeros/química , Dendrímeros/farmacologia , Glicopeptídeos/química , Glicopeptídeos/farmacologia , Lectinas/metabolismo , Pseudomonas aeruginosa/metabolismo , Humanos , Modelos Moleculares , Ligação Proteica , Infecções por Pseudomonas/microbiologiaRESUMO
Synthetic heteroglycoclusters are being subjected to increasing interest due to their potential to serve as selective ligands for carbohydrate-binding proteins. In this paper, we describe an expedient strategy to prepare cyclopeptides displaying well-defined distributions and combinations of carbohydrates. By using both oxime ligation and copper(I)-catalyzed alkyne-azide cycloaddition, two series of compounds bearing binary combinations of αMan, αFuc or ßLac in an overall tetravalent presentation, and either 2:2 or 3:1 relative proportions, have been prepared.
RESUMO
Synthetic glycoclusters and their related biological applications have stimulated increasing interest over the last decade. As a prerequisite to discovering active and selective therapeuticals, the development of multivalent glycoconjugates with diverse topologies is faced with inherent synthetic and structural characterisation difficulties. Here we describe a new series of molecularly-defined glycoclusters that were synthesized in a controlled manner using a robust and versatile divergent protocol. Starting from a Regioselectively Addressable Functionalized Template (RAFT) carrier, either a polylysine dendritic framework or a second RAFT, then 16 copies of ßGal, αMan, ßLac or cancer-related Thomsen-Freidenreich (αTF) antigen were successively conjugated within the same molecule using oxime chemistry. We thus obtained a new generation of dendri-RAFTs glycoclusters with high glycosidic density and variable spatial organizations. These compounds displaying 16 endgroups were unambiguously characterized by NMR spectroscopy and mass spectrometry. Further biological assays between a model lectin from Canavalia ensiformis (ConA) and mannosylated glycoclusters revealed a higher inhibition potency than the tetravalent counterpart, in particular for the hexadecavalent polylysine skeleton. Together with the efficiency of the synthetic and characterisation processes, this preliminary biological study provided clear evidence of promising properties that make the second generation of cyclopeptide-based glycoclusters attractive for biomedical applications.
Assuntos
Carboidratos/química , Peptídeos Cíclicos/síntese química , Canavalia/química , Modelos Moleculares , Estrutura MolecularRESUMO
Protein (lectin/toxin)-glycan interaction can be clinically harmful so that the design of inhibitors has become an aim. Cyclic decapeptides are suited as rigid carriers for carbohydrate derivatives. We herein document the bioactivity of sugar headgroups covalently attached to this carrier for the cases of five proteins, i.e. a potent biohazardous plant agglutinin, a leguminous model lectin and three adhesion/growth-regulatory human lectins. They represent the different types of topological organization within the galectin family. The relative inhibitory activities of glycoclusters with the three ligands (galactose, lactose and the disaccharide of the Thomsen-Friedenreich antigen) reflected the affinity of free carbohydrates, hereby excluding an impairment of binding activity by chemical derivatization and conjugation. Headgroup tailoring is thus one route to optimize activity and selectivity of cyclopeptide-based glycoclusters. The increase of ligand density from tetra- to hexadecavalency added a second route. The plant toxin and tandem-repeat-type galectin-4 were especially sensitive to this parameter change. Strategically combining solid-phase assays for screening with analysis of lectin binding to cells in different systems revealed efficient inhibition by distinct glycoclusters, thereby protecting cells from lectin association. Cyclic neoglycodecapeptides thus warrant further study as lectin-directed pharmaceuticals.
Assuntos
Assialoglicoproteínas/química , Glicopeptídeos/farmacologia , Proteínas Imobilizadas/química , Lectinas/metabolismo , Peptídeos Cíclicos/farmacologia , alfa-Fetoproteínas/química , Animais , Antígenos Glicosídicos Associados a Tumores/química , Ligação Competitiva , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Fetuínas , Galactose/química , Glicopeptídeos/síntese química , Glicopeptídeos/química , Humanos , Hidroxilaminas/síntese química , Lactose/química , Lectinas/antagonistas & inibidores , Lectinas/toxicidade , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/químicaRESUMO
Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.