Lectin LecA Research Articles

Biologically Active Heteroglycoclusters Constructed on a Pillar[5]arene-Containing [2]Rotaxane Scaffold.

A synthetic approach combining recent concepts for the preparation of multifunctional nanomolecules (click chemistry on multifunctional scaffolds) with supramolecular chemistry (self‐assembly to prepare rotaxanes) gave easy access to a large variety of sophisticated [2]rotaxane heteroglycoclusters. Specifically, compounds combining galactose and fucose have been prepared to target the two bacterial lectins (LecA and LecB) from the opportunistic pathogen Pseudomonas aeruginosa.

Structural Insight into Multivalent Galactoside Binding to Pseudomonas aeruginosa Lectin LecA

Multivalent galactosides inhibiting Pseudomonas aeruginosa biofilms may help control this problematic pathogen. To understand the binding mode of tetravalent glycopeptide dendrimer GalAG2 [(Gal-β-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2] to its target lectin LecA, crystal structures of LecA complexes with divalent analog GalAG1 [(Gal-β-OC6H4CO-Lys-Pro-Leu)2Lys-Phe-Lys-Ile-NH2] and related glucose-triazole linked bis-galactosides 3u3 [Gal-β-O(CH2)n-(C2HN3)-4-Glc-β-(C2HN3)-[β-Glc-4-(N3HC2)]2-(CH2)n-O-β-Gal (n = 1)] and 5u3 (n = 3) were obtained, revealing a chelate bound 3u3, cross-linked 5u3, and monovalently bound GalAG1. Nevertheless, a chelate bound model better explaining their strong LecA binding and the absence of lectin aggregation was obtained by modeling for all three ligands. A model of the chelate bound GalAG2·LecA complex was also obtained rationalizing its unusually tight LecA binding (KD = 2.5 nM) and aggregation by lectin cross-linking. The very weak biofilm inhibition with divalent LecA inhibitors suggests that lectin aggregation is necessary for biofilm inhibition by GalAG2, pointing to multivalent glycoclusters as a unique opportunity to control P. aeruginosa biofilms.

Pseudomonas Aeruginosa Lectins As Targets for Novel Antibacterials

Pseudomonas aeruginosa is one of the most widespread and troublesome opportunistic pathogens that is capable of colonizing various human tissues and organs and is often resistant to many currently used antibiotics. This resistance is caused by different factors, including the acquisition of specific resistance genes, intrinsic capability to diminish antibiotic penetration into the bacterial cell, and the ability to form biofilms. This situation has prompted the development of novel compounds differing in their mechanism of action from traditional antibiotics that suppress the growth of microorganisms or directly kill bacteria. Instead, these new compounds should decrease the pathogens' ability to colonize and damage human tissues by inhibiting the virulence factors and biofilm formation. The lectins LecA and LecB that bind galactose and fucose, as well as oligo- and polysaccharides containing these sugars, are among the most thoroughly-studied targets for such novel antibacterials. In this review, we summarize the results of experiments highlighting the importance of these proteins for P. aeruginosa pathogenicity and provide information on existing lectins inhibitors and their effectiveness in various experimental models. Particular attention is paid to the effects of lectins inhibition in animal models of infection and in clinical practice. We argue that lectins inhibition is a perspective approach to combating P. aeruginosa. However, despite the existence of highly effective in vitro inhibitors, further experiments are required in order to advance these inhibitors into pre-clinical studies.

Functionalization of a Rigid Divalent Ligand for LecA, a Bacterial Adhesion Lectin.

The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P. aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer–protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC).

Thiourea-based spacers in potent divalent inhibitors of Pseudomonas aeruginosa virulence lectin LecA.

A new divalent highly potent inhibitor of the Pseudomonas aeruginosa lectin and virulence factor LecA was prepared. It contains two thiourea linkages which were found to be in the Z,Z isomeric form. This brings the spacer into an elongated conformation required to bridge the two binding sites, which results in the chelating binding mode responsible for the high potency.

Expeditive synthesis of trithiotriazine-cored glycoclusters and inhibition of Pseudomonas aeruginosa biofilm formation.

Readily accessible, low-valency glycoclusters based on a triazine core bearing D-galactose and L-fucose epitopes are able to inhibit biofilm formation by Pseudomonas aeruginosa. These multivalent ligands are simple to synthesize, are highly soluble, and can be either homofunctional or heterofunctional. The galactose-decorated cluster shows good affinity for Pseudomonas aeruginosa lectin lecA. They are convenient biological probes for investigating the roles of lecA and lecB in biofilm formation.

A lipid zipper triggers bacterial invasion.

Glycosphingolipids are important structural constituents of cellular membranes. They are involved in the formation of nanodomains ("lipid rafts"), which serve as important signaling platforms. Invasive bacterial pathogens exploit these signaling domains to trigger actin polymerization for the bending of the plasma membrane and the engulfment of the bacterium--a key process in bacterial uptake. However, it is unknown whether glycosphingolipids directly take part in the membrane invagination process. Here, we demonstrate that a "lipid zipper," which is formed by the interaction between the bacterial surface lectin LecA and its cellular receptor, the glycosphingolipid Gb3, triggers plasma membrane bending during host cell invasion of the bacterium Pseudomonas aeruginosa. In vitro experiments with Gb3-containing giant unilamellar vesicles revealed that LecA/Gb3-mediated lipid zippering was sufficient to achieve complete membrane engulfment of the bacterium. In addition, theoretical modeling elucidated that the adhesion energy of the LecA-Gb3 interaction is adequate to drive the engulfment process. In cellulo experiments demonstrated that inhibition of the LecA/Gb3 lipid zipper by either lecA knockout, Gb3 depletion, or application of soluble sugars that interfere with LecA binding to Gb3 significantly lowered P. aeruginosa uptake by host cells. Of note, membrane engulfment of P. aeruginosa occurred independently of actin polymerization, thus corroborating that lipid zippering alone is sufficient for this crucial first step of bacterial host-cell entry. Our study sheds new light on the impact of glycosphingolipids in the cellular invasion of bacterial pathogens and provides a mechanistic explication of the initial uptake processes.

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 (Kd = 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.

Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA‐IL) by atomic force microscopy: influence of the glycocluster concentration

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA-IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA-IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra-galactosylated glycocluster and the PA-IL lectin for high concentration of lectins (2.5 μM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one-dimensional elongated aggregates to two-dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction.

Structure‐Based Optimization of the Terminal Tripeptide in Glycopeptide Dendrimer Inhibitors of Pseudomonas aeruginosa Biofilms Targeting LecA

The galactopeptide dendrimer GalAG2 ((β-Gal-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2) binds strongly to the Pseudomonas aeruginosa (PA) lectin LecA, and it inhibits PA biofilms, as well as disperses already established ones. By starting with the crystal structure of the terminal tripeptide moiety GalA-KPL in complex with LecA, a computational mutagenesis study was carried out on the galactotripeptide to optimize the peptide-lectin interactions. 25 mutants were experimentally evaluated by a hemagglutination inhibition assay, 17 by isothermal titration calorimetry, and 3 by X-ray crystallography. Two of these tripeptides, GalA-KPY (dissociation constant (K(D))=2.7 μM) and GalA-KRL (K(D)=2.7 μM), are among the most potent monovalent LecA ligands reported to date. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition and dispersal compared to those of GalAG2, particularly G2KPY ((β-Gal-OC6H4CO-Lys-Pro-Tyr)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2). The possibility to retain and even improve the biofilm inhibition in several analogues of GalAG2 suggests that it should be possible to fine-tune this dendrimer towards therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition through amino acid substitutions.

Secondary sugar binding site identified for LecA lectin from Pseudomonas aeruginosa

The galactose-specific lectin LecA from Pseudomonas aeruginosa is a target for the development of new anti-infectious compounds. Sugar based molecules with anti-adhesive properties present great potential in the fight against bacterial infection and biofilm formation. LecA is specific for oligosaccharides with terminal α-galactoside residues and displays strong affinity for melibiose (αGal1-6Glc) with a Kd of 38.8 µM. The crystal structure of LecA/melibiose complex shows classical calcium-bridged binding of αGal in the primary binding site but also revealed a secondary sugar binding site with glucose bound. This sugar binding site is in close proximity to the galactose binding one, is independent of calcium and mainly involves interactions with a symmetry-related protein. This discovery would help to the design of new potent inhibitors targeting both binding sites.

CH−π “T-Shape” Interaction with Histidine Explains Binding of Aromatic Galactosides to Pseudomonas aeruginosa Lectin LecA

The galactose specific lectin LecA mediates biofilm formation in the opportunistic pathogen P. aeruginosa . The interaction between LecA and aromatic β-galactoside biofilm inhibitors involves an intermolecular CH-π T-shape interaction between C(ε1)-H of residue His50 in LecA and the aromatic ring of the galactoside aglycone. The generality of this interaction was tested in a diverse family of β-galactosides. LecA binding to aromatic β-galactosides (KD ∼ 8 μM) was consistently stronger than to aliphatic β-galactosides (KD ∼ 36 μM). The CH-π interaction was observed in the X-ray crystal structures of six different LecA complexes, with shorter than the van der Waals distances indicating productive binding. Related XH/cation/π-π interactions involving other residues were identified in complexes of aromatic glycosides with a variety of carbohydrate binding proteins such as concanavalin A. Exploiting such interactions might be generally useful in drug design against these targets.

Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-β-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.

Efficient synthesis of phenylene-ethynylene rods and their use as rigid spacers in divalent inhibitors

The synthesis of phenylene-ethynylene rods and their use as rigid spacers is described. Alternation of a Sonogashira reaction and silyl group cleavage was used to obtain rigid spacers with even and odd numbers of phenylene units. Preliminary applications of these rods in divalent systems are shown. Inhibition studies with Pseudomonas Aeruginosa lectin LecA showed that the rigid spacer proved greatly beneficial for the inhibitory potency.

Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors

Synthetic glycopeptide dendrimers composed of a branched oligopeptide tree structure appended with glycosidic groups at its multiple N-termini were investigated for binding to the Pseudomonas aeruginosa lectins LecB and LecA. These lectins are partly responsible for the formation of antibiotic resistant biofilms in the human pathogenic bacterium P. aeruginosa, which causes lethal airway infections in immune-compromised and cystic fibrosis patients. Glycopeptide dendrimers with high affinity to the lectins were identified by screening of combinatorial libraries. Several of these dendrimers, in particular the LecB specific glycopeptide dendrimers FD2 and D-FD2 and the LecA specific glycopeptide dendrimers GalAG2 and GalBG2, also efficiently block P. aeruginosa biofilm formation and induce biofilm dispersal in vitro. Structure-activity relationship and structural studies are reviewed, in particular the observation that multivalency is essential to the anti-biofilm effect in these dendrimers.

ChemInform Abstract: Peptide and Glycopeptide Dendrimer Apple Trees as Enzyme Models and for Biomedical Applications.

AbstractReview: 84 refs.

Potent divalent inhibitors with rigid glucose click spacers for Pseudomonas aeruginosa lectin LecA

The synthesis of a new rigid spacer based on carbohydrate-triazole repeating units and their incorporation into divalent systems is described. Inhibition studies showed that a well-matched system with a rigid spacer with flexible ends leads to the most potent inhibition of Pseudomonas aeruginosa lectin LecA.

Peptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications

Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ~ 5,000 Da). Larger peptide dendrimers (MW ~ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat) ~ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobial peptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.

A Glycopeptide Dendrimer Inhibitor of the Galactose‐Specific Lectin LecA and of Pseudomonas aeruginosa Biofilms

Biofilm inhibition is achieved with a phenylgalactosyl peptide dendrimer (see picture) that binds to the galactose-specific lectin LecA of P. aeruginosa. The multivalency of the ligands is critical for biofilm inhibition, although the nature of the linker between the peptide dendrimer and the galactose can provide additional contacts to the lectin and also has an effect on the interaction.

AFM investigation of Pseudomonas aeruginosa lectin LecA (PA-IL) filaments induced by multivalent glycoclusters

Atomic force microscopy reveals that Pseudomonas aeruginosa LecA (PA-IL) and a tetra-galactosylated 1,3-alternate calix[4]arene-based glycocluster self-assemble according to an aggregative chelate binding mode to create monodimensional filaments. Lectin oligomers are identified along the filaments and defects in chelate binding generate branches and bifurcations. A molecular model with alternate 90° orientation of LecA tetramers is proposed to describe the organisation of lectins and glycoclusters in the filaments.