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.     

Combining glycomimetic and multivalent strategies toward designing potent bacterial lectin inhibitors

As part of ongoing activities toward the design of potent and selective ligands against galactoside-binding proteins from animal, bacterial, and plant lectins, a systematic investigation involving the synthesis and binding evaluations of a series of original β-C-galactopyranoside mimetics is described. The multivalent presentation of partly optimized candidates on various dendritic scaffolds through Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAc) has also been achieved. Biophysical investigations based on isothermal titration calorimetry (ITC) have indicated a dissociation constant in the low micromolar range for the best optimized monovalent conjugate (K(d)=37 μM). The results thus confirmed that stable C-galactosides could represent efficient synthetic glycomimetics of natural α-linked oligosaccharidic inhibitors of PA-IL lectin (Lec A) from the pathogenic Pseudomonas aeruginosa. Striking enhancements in the avidity of the glycoconjugates were also observed for tri-, hexa-, and nonavalent derivatives, among which the most potent exhibited dissociation constants below 500 nM, corresponding to a 400-fold increase in affinity compared with the β-D-Gal-O-Me used as reference. To deepen our understanding of the binding mode of the best glycomimetics involved in the recognition process, molecular modeling studies, docking calculations, and NMR diffusion measurements have been performed. Although favorable complementary interactions induced by the addition of the hydrophobic aglycon might explain the affinity enhancement, experimental determination of the size and the topology of the multivalent conjugates further supported the formation of aggregative complexes as a major multivalent binding mode. This work represents a systematic and comprehensive study towards a thorough understanding of the protein-carbohydrate interactions involved in Pseudomonas aeruginosa infection, and as such should prove useful for the development of stable and optimized anti-adhesive agents.     

Multiplexed binding determination of seven glycoconjugates for Pseudomonas aeruginosa lectin I (PA-IL) using a DNA-based carbohydrate microarray

The binding of seven multivalent glycoconjugates displaying linear or antenna-like structures and different electronic environments were evaluated towards PA-IL on a DNA-based carbohydrate microarray. The affinity can be modulated by the charge and the topology of the galactosylated derivatives.     

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.     

On-bead synthesis and binding assay of chemoselectively template-assembled multivalent neoglycopeptides

The investigation of recognition events between carbohydrates and proteins, especially the control of how spatial factors and binding avidity are correlated in, remains a great interest for glycomics. Therefore, the development of efficient methods for the rapid evaluation of new ligands such as multivalent glycoconjugates is essential for diverse diagnostic or therapeutic applications. In this paper we describe the synthesis of chemoselectively-assembled multivalent neoglycopeptides and the subsequent recognition assay on a solid support. Aminooxylated carbohydrates (betaLac-ONH(2) 4, alphaGalNAc-ONH(2) 9 and alphaMan-ONH(2) 13) have been prepared as carbohydrate-based recognition elements and assembled as clusters onto a cyclopeptidic scaffold by an oxime-based strategy in solid phase. Further binding tests between lectins and beads of resin derivatized with neoglycopeptides displaying clustered lactoses, N-acetylgalactoses and mannoses (18-20) have shown specific recognition and enhanced affinity through multivalent interactions, suggesting that the local density of carbohydrate-based ligands at the bead surface is crucial to improve the interaction of proteins of weak binding affinity. This solid phase strategy involving both molecular assembly and biological screening provides a rapid and efficient tool for various applications in glycomics.     

Novel Approaches To Design Glycan-Based Antibacterial Inhibitors

The interactions between bacterial lectins and carbohydrates on the host cell surface can mediate bacterial adhesion, invasion, and immune evasion. Multivalency plays a key role in these binding events. However, additional molecular mechanisms greatly impact multivalent binding recognition. To develop specific and effective bacterial inhibitors, a deeper understanding of the complex underlying mechanisms of bacterial adhesion processes is necessary. By interfering with bacterial adhesion, synthetic multivalent glycoconjugates do not only have the potential to improve or replace antibiotic treatments, but also represent useful tools to study carbohydrate-pathogen interactions. In this review, we highlight a few recent advances in the synthesis and application of synthetic glycan-based scaffolds to uncover the nature of glycan-bacteria interactions and to design efficient bacterial inhibitors.     

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.     

Membrane Deformation by Neolectins with Engineered Glycolipid Binding Sites

Lectins are glycan‐binding proteins that are involved in the recognition of glycoconjugates at the cell surface. When binding to glycolipids, multivalent lectins can affect their distribution and alter membrane shapes. Neolectins have now been designed with controlled number and position of binding sites to decipher the role of multivalency on avidity to a glycosylated surface and on membrane dynamics of glycolipids. A monomeric hexavalent neolectin has been first engineered from a trimeric hexavalent bacterial lectin, From this neolectin template, 13 different neolectins with a valency ranging from 0 to 6 were designed, produced, and analyzed for their ability to bind fucose in solution, to attach to a glycosylated surface and to invaginate glycolipid‐containing giant liposomes. Whereas the avidity only depends on the presence of at least two binding sites, the ability to bend and invaginate membranes critically depends on the distance between two adjacent binding sites.     

Multivalent Thiosialosides and Their Synergistic Interaction with Pathogenic Sialidases

Sialidases (SAs) hydrolyze sialyl residues from glycoconjugates of the eukaryotic cell surface and are virulence factors expressed by pathogenic bacteria, viruses, and parasites. The catalytic domains of SAs are often flanked with carbohydrate-binding module(s) previously shown to bind sialosides and to enhance enzymatic catalytic efficiency. Herein, non-hydrolyzable multivalent thiosialosides were designed as probes and inhibitors of V. cholerae, T. cruzi, and S. pneumoniae (NanA) sialidases. NanA was truncated from the catalytic and lectinic domains (NanA-L and NanA-C) to probe their respective roles upon interacting with sialylated surfaces and the synthetically designed di- and polymeric thiosialosides. The NanA-L domain was shown to fully drive NanA binding, improving affinity for the thiosialylated surface and compounds by more than two orders of magnitude. Importantly, each thiosialoside grafted onto the polymer was also shown to reduce NanA and NanA-C catalytic activity with efficiency that was 3000-fold higher than that of the monovalent thiosialoside reference. These results extend the concept of multivalency for designing potent bacterial and parasitic sialidase inhibitors.     

Solution and crystallographic studies of branched multivalent ligands that inhibit the receptor-binding of cholera toxin

The structure-based design of multivalent ligands offers an attractive strategy toward high affinity protein inhibitors. The spatial arrangement of the receptor-binding sites of cholera toxin, the causative agent of the severe diarrheal disease cholera and a member of the AB(5) bacterial toxin family, provides the opportunity of designing branched multivalent ligands with 5-fold symmetry. Our modular synthesis enabled the construction of a family of complex ligands with five flexible arms each ending with a bivalent ligand. The largest of these ligands has a molecular weight of 10.6 kDa. These ligands are capable of simultaneously binding to two toxin B pentamer molecules with high affinity, thus blocking the receptor-binding process of cholera toxin. A more than million-fold improvement over the monovalent ligand in inhibitory power was achieved with the best branched decavalent ligand. This is better than the improvement observed earlier for the corresponding nonbranched pentavalent ligand. Dynamic light scattering studies demonstrate the formation of concentration-dependent unique 1:1 and 1:2 ligand/toxin complexes in solution with no sign of nonspecific aggregation. This is in complete agreement with a crystal structure of the branched multivalent ligand/toxin B pentamer complex solved at 1.45 A resolution that shows the specific 1:2 ligand/toxin complex formation in the solid state. These results reiterate the power of the structure-based design of multivalent protein ligands as a general strategy for achieving high affinity and potent inhibition.     

Two-prong inhibitors for human carbonic anhydrase II

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.     

Precipitation-free high-affinity multivalent binding by inline lectin ligands

Multivalent ligand–protein interactions are a key concept in biology mediating, for example, signalling and adhesion. Multivalent ligands often have tremendously increased binding affinities. However, they also can cause crosslinking of receptor molecules leading to precipitation of ligand–receptor complexes. Plaque formation due to precipitation is a known characteristic of numerous fatal diseases limiting a potential medical application of multivalent ligands with a precipitating binding mode. Here, we present a new design of high-potency multivalent ligands featuring an inline arrangement of ligand epitopes with exceptionally high binding affinities in the low nanomolar range. At the same time, we show with a multi-methodological approach that precipitation of the receptor is prevented. We distinguish distinct binding modes of the ligands, in particular we elucidate a unique chelating binding mode, where four receptor binding sites are simultaneously bridged by one multivalent ligand molecule. The new design concept of inline multivalent ligands, which we established for the well-investigated model lectin wheat germ agglutinin, has great potential for the development of high-potency multivalent inhibitors as future therapeutics.

Integration of sugar epitopes into a backbone structure generates multivalent lectin ligands with a defined binding mode and high affinity without precipitating the protein.     

On the nature of the multivalency effect: a thermodynamic model

A quantitative model is proposed for the analysis of the thermodynamic parameters of multivalent interactions in dilute solutions or with immobilized multimeric receptor. The model takes into account all bound species and describes multivalent binding via two microscopic binding energies corresponding to inter- and intramolecular interactions (Delta G(o)inter and Delta G(o)intra), the relative contributions of which depend on the distribution of complexes with different numbers of occupied binding sites. The third component of the overall free energy, which we call the "avidity entropy" term, is a function of the degeneracy of bound states, Omega(i), which is calculated on the basis of the topology of interaction and the distribution of all bound species. This term grows rapidly with the number of receptor sites and ligand multivalency, it always favors binding, and explains why multivalency can overcome the loss of conformational entropy when ligands displayed at the ends of long tethers are bound. The microscopic parameters and may be determined from the observed binding energies for a set of oligovalent ligands by nonlinear fitting with the theoretical model. Here binding data obtained from two series of oligovalent carbohydrate inhibitors for Shiga-like toxins were used to verify the theory. The decavalent and octavalent inhibitors exhibit subnanomolar activity and are the most active soluble inhibitors yet seen that block Shiga-like toxin binding to its native receptor. The theory developed here in conjunction with our protocol for the optimization of tether length provides a predictive approach to design and maximize the avidity of multivalent ligands.     

Ferrocene-mannose conjugates as electrochemical molecular sensors for concanavalin A lectin

The binding affinity of a series of electroactive glycoconjugates, based on a ferrocene core bearing alpha-mannose units on one or both of its cyclopentadienyl rings, to lectin Con A was studied by isothermal titration calorimetry (ITC) and voltammetry. Voltammetric measurements were performed by differential pulse adsorptive stripping voltammetry (DPAdSV). Upon complexation of ferrocene-mannose conjugates with Con A, voltammograms showed a decrease of the peak current. Both the monomannosylated ferrocene and the bis(mannosylated) ferrocene derivatives form more stable complexes with Con A than methyl alpha-D-mannopyranoside. Bis(mannosylated) ferrocene conjugates were found to bind to Con A with enhanced affinity due to the multivalent effect. A comparison of the thermodynamic data obtained by ITC and voltammetry is presented.     

Structure-based virtual screening for novel potential selective inhibitors of class IIa histone deacetylases for cancer treatment

The fundamental cause of human cancer is strongly influenced by down- or up-regulations of epigenetic factors. Upregulated histone deacetylases (HDAC) have been shown to be effectively neutralized by the action of HDACs inhibitors (HDACi). However, cytotoxicity has been reported in normal cells because of non-specificity of several available HDACis that are in clinical use or at different phases of clinical trials. Because of the high amino acid sequence and structural similarity among HDAC enzymes, it is believed to be a challenging task to obtain isoform-selectivity. The essential aim of the present research work was to identify isoform-selective inhibitors against class IIa HDACs via structure-based drug design. Based on the highest binding affinity and isoform-selectivity, the top-ranked inhibitors were in silico tested for their absorption, distribution, metabolism, elimination, and toxicity (ADMET) properties, which were classified as drug-like compounds. Later, molecular dynamics simulation (MD) was carried out for all compound-protein complexes to evaluate the structural stability and the biding mode of the inhibitors, which showed high stability throughout the 100 ns simulation. Free binding energy predictions by MM-PBSA method showed the high binding affinity of the identified compounds toward their respective targets. Hence, these inhibitors could be used as drug candidates or as lead compounds for more in silico or in vitro optimization to design safe isoform-selective HDACs inhibitors.     

Multivalent Effect in Glycosidase Inhibition: The End of the Beginning

Glycosidases are ubiquitous enzymes involved in a diversity of key biological processes such as energy uptake or cell wall degradation. The design of specific glycosidase inhibitors has been therefore the subject of intense research efforts in academia and pharmaceutical industry. However, until recently, the study of the impact of multivalency on glycosidase inhibition was almost completely neglected. The following account will review our ten year journey on the design of multivalent glycomimetics within our research group, from the discovery of the first strong multivalent effect in glycosidase inhibition to the high‐resolution crystal structures of Jack bean α‐mannosidase in complex with the multimeric inhibitor displaying the largest binding enhancements reported so far.     

The multivalent effect in glycosidase inhibition: probing the influence of architectural parameters with cyclodextrin-based iminosugar click clusters

In contrast to most lectins, glycosidases may appear to be unpromising targets for multivalent binding because they display only a single active site. To explore the potential of multivalency on glycosidase inhibition, unprecedented cyclodextrin-based iminosugar conjugates have been designed and prepared. The synthesis was performed by way of Cu(I) -catalyzed azide-alkyne cycloaddition reaction under microwave activation between propargylated multivalent β-cyclodextrins and an azide-armed N-alkyl 1-deoxynojirimycin derivative. Evaluation with a panel of glycosidases of this new class of glycomimetic clusters revealed the strongest affinity enhancement observed to date for a multivalent glycosidase inhibitor, with binding enhancement up to four orders of magnitude over the corresponding monovalent ligand for α-mannosidase. These results demonstrate that the multivalency concept extends beyond carbohydrate-lectin recognition processes to glycomimetic-enzyme inhibition.     

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.     

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers

Divalent precision glycooligomers terminating in N‐acetylneuraminic acid (Neu5Ac) or 3′‐sialyllactose (3′‐SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co‐crystallization studies with the sialic acid‐binding major capsid protein VP1 of human Trichodysplasia spinulosa‐associated Polyomavirus. High‐resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.     

Optimization of bivalent glutathione S-transferase inhibitors by combinatorial linker design

Dimeric glutathione S-transferases (GSTs) are pharmacological targets for several diseases, including cancer. Isoform specificity has been difficult to achieve due to their overlapping substrate selectivity. Here we demonstrate the utility of bivalent GST inhibitors and their optimization via combinatorial linker design. A combinatorial library with dipeptide linkers emanating symmetrically from a central scaffold (bis-3,5-aminomethyl benzoic acid, AMAB) to connect two ethacrynic acid moieties was prepared and decoded via iterative deconvolution, against the isoforms GSTA1-1 and GSTP1-1. The library yielded high affinity GSTA1-1 selective inhibitors (70-120-fold selectivity) and with stoichiometry of one inhibitor: one GSTA1-1 dimer. Saturation Transfer Difference (STD) NMR with one of these inhibitors, with linker structure (Asp-Gly-AMAB-Gly-Asp) and K(D) = 42 nM for GSTA1-1, demonstrates that the Asp-Gly linker interacts tightly with GSTA1-1, but not P1-1. H/D exchange mass spectrometry was used to map the protein binding site and indicates that peptides within the intersubunit cleft and in the substrate binding site are protected by inhibitor from solvent exchange. A model is proposed for the binding orientation of the inhibitor, which is consistent with electrostatic complementarity between the protein cleft and inhibitor linker as the source of isoform selectivity and high affinity. The results demonstrate the utility of combinatorial, or "irrational", linker design for optimizing bivalent inhibitors.