Multivalent gold glycoclusters: high affinity molecular recognition by bacterial lectin PA-IL

Multivalent protein-carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin-carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate-functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA-IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000-fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K(d)) of 50 nM per monosaccharide, the most effective ligand of PA-IL measured to date; another demonstration of the potential of glyco-nanotechnology towards multivalent tools and potent anti-adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Glycopeptide Mimetics Recapitulate High‐Mannose‐Type Oligosaccharide Binding and Function

High‐mannose‐type glycans (HMTGs) decorating viral spike proteins are targets for virus neutralization. For carbohydrate‐binding proteins, multivalency is important for high avidity binding and potent inhibition. To define the chemical determinants controlling multivalent interactions we designed glycopeptide HMTG mimetics with systematically varied mannose valency and spacing. Using the potent antiviral lectin griffithsin (GRFT) as a model, we identified by NMR spectroscopy, SPR, analytical ultracentrifugation, and microcalorimetry glycopeptides that fully recapitulate the specificity and kinetics of binding to Man₉GlcNAc₂Asn and a synthetic nonamannoside. We find that mannose spacing and valency dictate whether glycopeptides engage GRFT in a face‐to‐face or an intermolecular binding mode. Surprisingly, although face‐to‐face interactions are of higher affinity, intermolecular interactions are longer lived. These findings yield key insights into mechanisms involved in glycan‐mediated viral inhibition.     

High-affinity chelator thiols for switchable and oriented immobilization of histidine-tagged proteins: a generic platform for protein chip technologies

Protein micro-/nanoarrays are becoming increasingly important in systematic approaches for the exploration of protein-protein interactions and dynamic protein networks, so there is a high demand for specific, generic, stable, uniform, and locally addressable protein immobilization on solid supports. Here we present multivalent metal-chelating thiols that are suitable for stable binding of histidine-tagged proteins on biocompatible self-assembled monolayers (SAMs). The architectures and physicochemical properties of these SAMs have been probed by various surface-sensitive techniques such as contact angle goniometry, ellipsometry, and infrared reflection-absorption spectroscopy. The specific molecular organization of proteins and protein complexes was demonstrated by surface plasmon resonance, confocal laser scanning, and atomic force microscopy. In contrast to the mono-NTA/His6 tag interaction, which has major drawbacks because of its low affinity and fast dissociation, drastically improved stability of protein binding by these multivalent chelator surfaces was observed. The immobilized histidine-tagged proteins are uniformly oriented and retain their function. At the same time, proteins can be removed from the chip surface under mild conditions (switchability). This new platform for switchable and oriented immobilization should assist proteome-wide wide analyses of protein-protein interactions as well as structural and single-molecule studies.     

Multivalent Gold Glycoclusters: High Affinity Molecular Recognition by Bacterial Lectin PA‐IL

Multivalent protein‐carbohydrate interactions are involved in the initial stages of many fundamental biological and pathological processes through lectin–carbohydrate binding. The design of high affinity ligands is therefore necessary to study, inhibit and control the processes governed through carbohydrate recognition by their lectin receptors. Carbohydrate‐functionalised gold nanoclusters (glyconanoparticles, GNPs) show promising potential as multivalent tools for studies in fundamental glycobiology research as well as biomedical applications. Here we present the synthesis and characterisation of galactose functionalised GNPs and their effectiveness as binding partners for PA‐IL lectin from Pseudomonas aeruginosa. Interactions were evaluated by hemagglutination inhibition (HIA), surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) assays. Results show that the gold nanoparticle platform displays a significant cluster glycoside effect for presenting carbohydrate ligands with almost a 3000‐fold increase in binding compared with a monovalent reference probe in free solution. The most effective GNP exhibited a dissociation constant (K_d) of 50 nM per monosaccharide, the most effective ligand of PA‐IL measured to date; another demonstration of the potential of glyco‐nanotechnology towards multivalent tools and potent anti‐adhesives for the prevention of pathogen invasion. The influence of ligand presentation density on their recognition by protein receptors is also demonstrated.     

Fullerene‐sp2‐Iminosugar Balls as Multimodal Ligands for Lectins and Glycosidases: A Mechanistic Hypothesis for the Inhibitory Multivalent Effect

Concerted functioning of lectins and carbohydrate‐processing enzymes, mainly glycosidases, is essential in maintaining life. It was commonly assumed that the mechanisms by which each class of protein recognizes their cognate sugar partners are intrinsically different: multivalency is a characteristic feature of carbohydrate–lectin interactions, whereas glycosidases bind to their substrates or substrate‐analogue inhibitors in monovalent form. Recent observations on the glycosidase inhibitory potential of multivalent glycomimetics have questioned this paradigm and led to postulate an inhibitory multivalent effect. Here the mechanisms at the origin of this phenomenon have been investigated. A D ‐gluco‐configured sp²‐iminosugar glycomimetic motif, namely 1‐amino‐5N,6O‐oxomethylydenenojirimycin (1N‐ONJ), behaving, simultaneously, as a ligand of peanut agglutinin (PNA) lectin and as an inhibitor of several glycosidases, has been identified. Both the 1N‐ONJ–lectin‐ and 1N‐ONJ–glycosidase‐recognition processes have been found to be sensitive to multivalency, which has been exploited in the design of a lectin–glycosidase competitive assay to explore the implication of catalytic and non‐glycone sites in enzyme binding. A set of isotropic dodecavalent C₆₀‐fullerene–sp²‐iminosugar balls incorporating matching or mismatching motifs towards several glycosidases (inhitopes) was synthesized for that purpose, thereby preventing differences in binding modes arising from orientational preferences. The data supports that: 1) multivalency allows modulating the affinity and selectivity of a given inhitope towards glycosidases; 2) multivalent presentation can switch on the inhibitory capacity for some inhitope–glycosidase pairs, and 3) interactions of the multivalent inhibitors with non‐glycone sites is critical for glycosidase recognition. The ensemble of results point to a shift in the binding mode on going from monovalent to multivalent systems: in the first case a typical ′′key–lock′′ model involving, essentially, the high‐affinity active site can be assumed, whereas in the second, a lectin‐like behavior implying low‐affinity non‐glycone sites probably operates. The differences in responsiveness to multivalency for different glycosidases can then be rationalized in terms of the structure and accessibility of the corresponding carbohydrate‐binding regions.     

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.     

Regioselective 3-O-Substitution of Unprotected Thiodigalactosides: Direct Route to Galectin Inhibitors

The synthesis of tailored bioactive carbohydrates usually comprises challenging (de)protection steps, which lowers synthetic yields and increases time demands. We present here a regioselective single-step introduction of benzylic substituents at 3-hydroxy groups of β-d -galactopyranosyl-(1→1)-thio-β-d -galactopyranoside (TDG) employing dibutyltin oxide in good yields. These glycomimetics act as inhibitors of galectins—human lectins, which are biomedically attractive targets for therapeutic inhibition in, for example, cancerogenesis. The affinity of the prepared glycomimetics to galectin-1 and galectin-3 was studied in enzyme-linked immunosorbent (ELISA)-type assays and their potential to inhibit galectin binding on the cell surface was shown. We used our original in vivo biotinylated galectin constructs for easy detection by flow cytometry. The results of the biological experiments were compared with data from molecular modeling with both galectins. The present work reveals a facile and elegant synthetic route for the preparation of TDG-derived glycomimetics that exhibit differing selectivity and affinity to galectins depending on the choice of 3-O-substitution.     

Sequence‐Defined Introduction of Hydrophobic Motifs and Effects in Lectin Binding of Precision Glycomacromolecules

This study investigates the influence of an increasingly hydrophobic backbone of multivalent glycomimetics based on sequence‐defined oligo(amidoamines) on their resulting affinity toward bacterial lectins. Glycomacromolecules are obtained by stepwise assembly of tailor‐made building blocks on solid support, using both hydrophobic aliphatic and aromatic building blocks to enable a gradual change in hydrophobicity of the backbone. Their binding behavior toward model lectin Concanavalin A (ConA) is evaluated using isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) showing higher affinities for glycomacromolecules with higher content of hydrophobic and aromatic moieties in the backbone. Finally, glycomacromolecules are tested in a bacterial adhesion inhibition study against Escherichia coli where more hydrophobic backbones yield higher inhibitory potentials most likely due to additional secondary interactions with hydrophobic regions of the protein receptor as well as a change in conformation exposing carbohydrate ligands for increased binding. Overall, the results highlight the influence and thereby importance of the polymer backbone itself on the resulting properties of polymeric biomimetics.     

α-N-Linked glycopeptides: conformational analysis and bioactivity as lectin ligands

Natural N-glycosylation involves a β-anomeric linkage connecting the sugar to one asparagine residue of the protein. We herein report NMR- and modelling-based data on glycomimetics containing α-glycosidic linkages. The bioactivity of α-Gal-containing glycopeptides has been documented by revealing binding to two plant lectins, i.e. a potent β-trefoil toxin (Viscum album agglutinin) and β-sandwich lectin (Erythrina corallodendron agglutinin), by NMR protocols. Docking provided insights into the 3D structures of the resulting complexes. These results provide the basis to introduce α-substituted neoglycopeptides to the toolbox of scaffold for the design of potent lectin inhibitors.     

Investigation of monovalent and bivalent enantioselective molecular recognition by electrospray ionization-mass spectrometry and tandem mass spectrometry

In this work is described the investigation of bivalent versus monovalent enantioselective molecular recognition in the context of enantioselective separations. Electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) are used for evaluating enantioselective systems through the measurement of (1) relative solution-phase binding constants via titration and (2) relative gas-phase binding via collision threshold dissociation. In HPLC, a cinchonane-type chiral stationary phase (CSP) based on tert.-butylcarbamoylquinine provides vastly increased retention and enantioselectivity for separation of bivalent versus monovalent alkoxy-benzoyl-N-blocked leucine enantiomers. The bivalent enantiomers are able to span and simultaneously interact with multiple interaction sites on the CSP surface, leading to enhanced separation. ESI-MS titration measurements also show an increased avidity for binding between bivalent selector and bivalent selectand, compared with the monovalent system. However, enhanced enantioselectivities measured in HPLC for the bivalent system cannot be reproduced by MS due to inherent mechanistic differences. Assumed discrepancies in relative response factors also give rise to systematic errors which are discussed. The results of MS/MS gas-phase experiments show that enantioselectivity is essentially lost in the absence of solvation, but that dissociation thresholds can provide a measure of relative dissociation energy in the bivalent interaction system compared to the monovalent counterpart. Such measurements may prove useful and efficient in better understanding multivalent interactions, in line with current theoretical considerations of effective concentrations and ion trap effects. This is the first application of mass spectrometric methods for assessing increased avidity of binding in multivalent enantioselective molecular recognition.     

Cinnamide Derivatives of d‐Mannose as Inhibitors of the Bacterial Virulence Factor LecB from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic Gram‐negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate‐based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug‐like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d‐mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20 derivatives with increased potency compared to the unsubstituted cinnamide. The structure–activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure‐based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half‐lives in the 5–20 min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti‐infectives against this multidrug‐resistant pathogen.     

Dynamic Cooperative Glycan Assembly Blocks the Binding of Bacterial Lectins to Epithelial Cells

Pathogens frequently rely on lectins for adhesion and cellular entry into the host. Since these interactions typically result from multimeric binding of lectins to cell‐surface glycans, novel therapeutic strategies are being developed with the use of glycomimetics as competitors of such interactions. Herein we study the benefit of nucleic acid based oligomeric assemblies with PNA–fucose conjugates. We demonstrate that the interactions of a lectin with epithelial cells can be inhibited with conjugates that do not form stable assemblies in solution but benefit from cooperativity between ligand–protein interactions and PNA hybridization to achieve high affinity. A dynamic dimeric assembly fully blocked the binding of the fucose‐binding lectin BambL of Burkholderia ambifaria , a pathogenic bacterium, to epithelial cells with an efficiency of more than 700‐fold compared to l ‐fucose.     

Achieving High Affinity towards a Bacterial Lectin through Multivalent Topological Isomers of Calix[4]arene Glycoconjugates

A family of seven topologically isomeric calix[4]arene glycoconjugates was prepared through the synthesis of a series of alkyne‐derivatised calix[4]arene precursors that are suitable for the attachment of sugar moieties by microwave‐assisted copper(I)‐catalysed azide–alkyne cycloaddition (CuAAC). The glycoconjugates thus synthesised comprised one mono‐functionalised derivative, two 1,2‐ or 1,3‐divalent regioisomers, one trivalent and three tetravalent topoisomers in the cone, partial cone or 1,3‐alternate conformations. The designed glycoconjugates were evaluated as ligands for the galactose‐binding lectin PA‐IL from the opportunistic bacterium Pseudomonas aeruginosa, a major causative agent of lung infections in cystic fibrosis patients. Binding affinities were determined by isothermal titration calorimetry (ITC), and the interaction with the lectin was shown to be strongly dependant on both the valence and the topology. Whereas the trivalent conjugate displayed enhanced affinity when compared to a monosaccharide model, the tetravalent conjugates are to‐date the highest‐affinity ligands measured by ITC. The topologies presenting carbohydrates on both faces of calixarene are the most potent ones with dissociation constants of approximately 200 nM . Molecular modelling suggests that such a multivalent molecule can efficiently chelate two of the binding sites of the tetrameric lectin; this explains the 800‐fold increase of affinity achieved by the tetravalent molecule. Surface plasmon resonance (SPR) experiments confirmed that this glycoconjugate is the strongest inhibitor for binding of PA‐IL to galactosylated surfaces for potential applications as an anti‐adhesive agent.     

In silico mutagenesis and docking studies of Pseudomonas aeruginosa PA-IIL lectin predicting binding modes and energies

This article is focused on the application of two types of docking software, AutoDock and DOCK. It is aimed at studying the interactions of a calcium-dependent bacterial lectin PA-IIL (from Pseudomonas aeruginosa) and its in silico mutants with saccharide ligands. The effect of different partial charges assigned to the calcium ions was tested and evaluated in terms of the best agreement with the crystal structure. The results of DOCK were further optimized by molecular dynamics and rescored using AMBER. For both software, the agreement of the docked structures and the provided binding energies were evaluated in terms of prediction accuracy. This was carried out by comparing the computed results to the crystal structures and experimentally determined binding energies, respectively. The performance of both docking software applied on a studied problem was evaluated as well. The molecular docking methods proved efficient in identifying the correct binding modes in terms of geometry and partially also in predicting the preference changes caused by mutation. Obtaining a reasonable in silico method for the prediction of lectin-saccharide interactions may be possible in the future.     

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.     

Considerations concerning interaction characterization of oligopeptide mixtures with vancomycin using affinity capillary electrophoresis-electrospray mass spectrometry.

In the past few years affinity capillary electrophoresis (ACE) has proven to be a powerful tool to study molecular interactions. In ACE the change in electrophoretic mobility between a free and a complexed ligand with a receptor dissolved in the background electrolyte is observed. It provides an accurate way to calculate binding or dissociation constants and, when coupled to mass spectrometry, it forms a promising method to analyze solution-based combinatorial libraries. We report a model study on the macrocyclic antibiotic vancomycin using a 36-component library of tetrapeptides of the type 9-fluorenylmethoxycarbonyl (Fmoc)-L-Asp-L-Asp-D-Xaa-D-Xaa. The mass spectrometry conditions were optimized by fine-tuning the background electrolyte and sheath flow composition to achieve optimal sensitivity in the negative ionization mode. Different types of capillaries were also evaluated on their potential to screen combinatorial libraries. The library components that show the strongest interaction were identified. The dissociation constants of a mixture of six compounds with a broad affinity range were simultaneously established by Scatchard analysis on ACE-MS.     

A LecA Ligand Identified from a Galactoside‐Conjugate Array Inhibits Host Cell Invasion by Pseudomonas aeruginosa

Lectin LecA is a virulence factor of Pseudomonas aeruginosa involved in lung injury, mortality, and cellular invasion. Ligands competing with human glycoconjugates for LecA binding are thus promising candidates to counteract P. aeruginosa infections. We have identified a novel divalent ligand from a focused galactoside(Gal)‐conjugate array which binds to LecA with very high affinity (K_d=82 nM ). Crystal structures of LecA complexed with the ligand together with modeling studies confirmed its ability to chelate two binding sites of LecA. The ligand lowers cellular invasiveness of P. aeruginosa up to 90 % when applied in the range of 0.05–5 μM . Hence, this ligand might lead to the development of drugs against P. aeruginosa infection.     

Self-assembled multivalent carbohydrate ligands

Materials that display multiple carbohydrate residues have gained much attention due to their potential to inhibit or modulate biological multivalent interactions. These materials can be grouped accordingly to the way they are prepared, as unimolecular or as self-assembled systems. Both systems take advantage of the fact that multivalent interactions have significantly higher binding affinity than the corresponding monovalent interactions. The self-assembled system is a more recent field of research compared to the unimolecular system. In this review, we describe current efforts to realize multivalent carbohydrate interactions from the perspective of synthetic self-assembled systems. We limit the scope to self-assembled systems that are stable, soluble in aqueous solution and morphologically discrete. We grouped them into two separate categories. In the first category carbohydrate ligands self-assemble onto a pre-organized nanostructure, and in the second carbohydrate-conjugated block molecules spontaneously assemble to construct morphologically distinct nanostructures.     

Synthesis and binding properties of divalent and trivalent clusters of the Lewis a disaccharide moiety to Pseudomonas aeruginosa lectin PA-IIL

The synthesis of oligomeric glycocomimetics has been performed for targeting the Pseudomonas aeruginosa PA-IIL lectin, which is of therapeutical interest for anti-adhesive treatment. The disaccharide alpha-L-Fucp-(1-->4)-beta-D-GlcNAc, which is a high-affinity ligand of the lectin, has been coupled to dimeric and trimeric linkers with various lengths and geometries. A series of linear dimers displayed an efficient clustering effect and a very strong affinity, with a lower dissociation constant of 90 nM. The trimeric compound was less efficient in inhibition assays but displayed high affinity in solution. Titration microcalorimetry and molecular modeling allowed in-depth analysis and rationalization of the binding data. These glycoclusters could act by crosslinking the lectins present on the surface of bacteria and therefore interfere with host recognition or biofilm formation.