Thiodigalactoside-Bovine Serum Albumin Conjugates as High-Potency Inhibitors of Galectin-3: An Outstanding Example of Multivalent Presentation of Small Molecule Inhibitors.

Galectin inhibitors are urgently needed to understand the mode of action and druggability of different galectins, but potent and selective agents still evade researchers. Small-sized inhibitors based on thiodigalactoside (TDG) have shown their potential while modifications at their C3 position indicated a strategy to improve selectivity and potency. Considering the role of galectins as glycoprotein traffic police, involved in multivalent bridging interactions, we aimed to create multivalent versions of the potent TDG inhibitors. We herein present for the first time the multivalent attachment of a TDG derivative using bovine serum albumin (BSA) as the scaffold. An efficient synthetic method is presented to obtain a novel type of neoglycosylated proteins loaded with different numbers of TDG moieties. A polyethylene glycol (PEG)-spacer is introduced between the TDG and the protein scaffold maintaining appropriate accessibility for an adequate galectin interaction. The novel conjugates were evaluated in galectin binding and inhibition studies in vitro. The conjugate with a moderate density of 19 conjugated TDGs was identified as one of the most potent multivalent Gal-3 inhibitors so far, with a clear demonstration of the benefit of a multivalent ligand presentation. The described method may facilitate the development of specific galectin inhibitors and their application in biomedical research.

KnowVolution Campaign of an Aryl Sulfotransferase Increases Activity toward Cellobiose.

Sulfated polysaccharides such as cellulose can mimic the functionalities of pathophysiologically important glycosaminoglycans. Enzymatic sulfation offers a green chemistry route to selective (mono)sulfation of oligosaccharides (e.g., cellobiose as a building block of cellulose) in aqueous solution, at ambient temperature, and high chemoselectivity. Here, we report the first KnowVolution campaign for the aryl sulfotransferase B (ASTB) from Desulfitobacterium hafniense to advance ASTB toward a synthetically attractive biocatalyst. The generated final recombination variant (ASTB-M5) carries two amino acid substitutions (Leu446Pro and Val579Lys) leading to an up to 7.6-fold increase in specific activity (6.15 U mg-1 ) that was obtained with one round of KnowVolution. Mass spectrometry analysis confirmed a monosulfated product of cellobiose and structure elucidation by NMR confirmed the sulfation at the positions C-3 or C-4 of GlcNAc-linker-tBoc as opposed to the preferred C-6 by chemical means. Computational analysis suggested an important role of Leu446Pro in substrate-binding and recognized Val579Lys as a distal substitution.

Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling.

Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with Kd reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N'-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum.

Tailored Multivalent Neo-Glycoproteins: Synthesis, Evaluation, and Application of a Library of Galectin-3-Binding Glycan Ligands.

Galectin-3 (Gal-3), a member of the ß-galactoside-binding lectin family, is a tumor biomarker and involved in tumor angiogenesis and metastasis. Gal-3 is therefore considered as a promising target for early cancer diagnosis and anticancer therapy. We here present the synthesis of a library of tailored multivalent neo-glycoproteins and evaluate their Gal-3 binding properties. By the combinatorial use of glycosyltransferases and chemo-enzymatic reactions, we first synthesized a set of N-acetyllactosamine (Galß1,4GlcNAc; LacNAc type 2)-based oligosaccharides featuring five different terminating glycosylation epitopes, respectively. Neo-glycosylation of bovine serum albumin (BSA) was accomplished by dialkyl squarate coupling to lysine residues resulting in a library of defined multivalent neo-glycoproteins. Solid-phase binding assays with immobilized neo-glycoproteins revealed distinct affinity and specificity of the multivalent glycan epitopes for Gal-3 binding. In particular, neo-glycoproteins decorated with N',N″-diacetyllactosamine (GalNAcß1,4GlcNAc; LacdiNAc) epitopes showed high selectivity and were demonstrated to capture Gal-3 from human serum with high affinity. Furthermore, neo-glycoproteins with terminal biotinylated LacNAc glycan motif could be utilized as Gal-3 detection agents in a sandwich enzyme-linked immunosorbent assay format. We conclude that, in contrast to antibody-based capture steps, the presented neo-glycoproteins are highly useful to detect functionally intact Gal-3 with high selectivity and avidity. We further gain novel insights into the binding affinity of Gal-3 using tailored multivalent neo-glycoproteins, which have the potential for an application in the context of cancer-related biomedical research.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands.

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galß1, 3GlcNAc) and type 2 (Galß1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant ß1,3-galactosyltransferase from Escherichia coli and ß1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.

High-sensitivity real-time analysis of nanoparticle toxicity in green fluorescent protein-expressing zebrafish.

Gold nanoparticles (AuNP) show great potential for diagnostic and therapeutic application in humans. A great number of studies have tested the cytotoxicity of AuNP using cell culture. There is, however, an urgent need to test AuNP in vertebrate animal models that interrogate biodistribution and complex biological traits like organ development, whole body metabolism, and cognitive function. The sheer number of different compounds precludes the use of small rodent model for initial screening. The extended fish embryo test (FET) is used here to bridge the gap between cell culture and small animal models. A study on the toxicity of ultrasmall AuNP in wild type and transgenic zebrafish is presented. FET faithfully reproduce all important findings of a previous study in HeLa cells and add new important information on teratogenicity and hepatotoxicity that could not be gained from studying cultured cells.

Enzymatic Synthesis of Glycans and Glycoconjugates.

Glycoconjugates have great potential to improve human health in a multitude of different ways and fields. Prominent examples are human milk oligosaccharides and glycosaminoglycans. The typical choice for the production of homogeneous glycoconjugates is enzymatic synthesis. Through the availability of expression and purification protocols, recombinant Leloir glycosyltransferases are widely applied as catalysts for the synthesis of a wide range of glycoconjugates. Extensive utilization of these enzymes also depends on the availability of activated sugars as building blocks. Multi-enzyme cascades have proven a versatile technique to synthesize and in situ regenerate nucleotide sugar.In this chapter, the functions and mechanisms of Leloir glycosyltransferases are revisited, and the advantage of prokaryotic sources and production systems is discussed. Moreover, in vivo and in vitro pathways for the synthesis of nucleotide sugar are reviewed. In the second part, recent and prominent examples of the application of Leloir glycosyltransferase are given, i.e., the synthesis of glycosaminoglycans, glycoconjugate vaccines, and human milk oligosaccharides as well as the re-glycosylation of biopharmaceuticals, and the status of automated glycan assembly is revisited.

Sequence-Defined Heteromultivalent Precision Glycomacromolecules Bearing Sulfonated/Sulfated Nonglycosidic Moieties Preferentially Bind Galectin-3 and Delay Wound Healing of a Galectin-3 Positive Tumor Cell Line in an In Vitro Wound Scratch Assay.

Within this work, a new class of sequence-defined heteromultivalent glycomacromolecules bearing lactose residues and nonglycosidic motifs for probing glycoconjugate recognition in carbohydrate recognition domain (CRD) of galectin-3 is presented. Galectins, a family of ß-galactoside-binding proteins, are known to play crucial roles in different signaling pathways involved in tumor biology. Thus, research has focused on the design and synthesis of galectin-targeting ligands for use as diagnostic markers or potential therapeutics. Heteromultivalent precision glycomacromolecules have the potential to serve as ligands for galectins. In this work, multivalency and the introduction of nonglycosidic motifs bearing either neutral, amine, or sulfonated/sulfated groups are used to better understand binding in the galectin-3 CRD. Enzyme-linked immunosorbent assays and surface plasmon resonance studies are performed, revealing a positive impact of the sulfonated/sulfated nonglycosidic motifs on galectin-3 binding but not on galectin-1 binding. Selected compounds are then tested with galectin-3 positive MCF 7 breast cancer cells using an in vitro would scratch assay. Preliminary results demonstrate a differential biological effect on MCF 7 cells with high galectin-3 expression in comparison to an HEK 293 control with low galectin-3 expression, indicating the potential for sulfonated/sulfated heteromultivalent glycomacromolecules to serve as preferential ligands for galectin-3 targeting.

Galectin-Carbohydrate Interactions in Biomedicine and Biotechnology.

Cellular communication events are mediated by interactions between cell-surface sugars and lectins, which are carbohydrate-binding proteins. Galectins are ß-galactosyl-binding lectins that bridge molecules by their sugar moieties, forming a signaling and adhesion network. Severe changes in glycosylation and galectin expression accompany major processes in oncogenesis, cardiovascular disorders, and other pathologies, making galectins attractive therapeutic targets. Here we discuss advanced strategies of chemo-enzymatic carbohydrate synthesis for creating lead glycomimetics and (neo-)glycoconjugates for galectin-1 and -3 targeting in biomedicine and biotechnology. We will describe the challenges and bottlenecks on the route into biomedical and biotechnological practice and present the first clinical candidates. The coming era will see an exciting translation of selective well-defined high-affinity galectin ligands from bench to bedside.

Effects of linker and liposome anchoring on lactose-functionalized glycomacromolecules as multivalent ligands for binding galectin-3.

In this work, we present a bottom-up approach for the synthesis of lactose-functionalized glycomacromolecules and glycofunctionalized liposomes and apply these compounds to investigate their effects of multivalent presentation on binding to galectin-3. Step-wise assembly of tailor-made building blocks on solid supports was used to synthesize a series of oligo(amidoamine) scaffolds that were further conjugated to lactose via copper catalyzed 1,3-dipolar cycloaddition. Binding studies with galectin-3 revealed affinities in the micromolar range that increased with increasing carbohydrate valency, and decreased with increasing size and linker flexibility. To further explore their multivalency, selected glycomacromolecules were conjugated to lipids and used in liposomal formulations. Binding studies show a further increase in binding in nanomolar ranges in dependence of both ligand structure and liposomal presentation, demonstrating the power of combining the two approaches.

Galectin Binding to Neo-Glycoproteins: LacDiNAc Conjugated BSA as Ligand for Human Galectin-3.

Carbohydrate-lectin interactions are relatively weak. As they play an important role in biological recognition processes, multivalent glycan ligands are designed to enhance binding affinity and inhibitory potency. We here report on novel neo-glycoproteins based on bovine serum albumin as scaffold for multivalent presentation of ligands for galectins. We prepared two kinds of tetrasaccharides (N-acetyllactosamine and N,N-diacetyllactosamine terminated) by multi-step chemo-enzymatic synthesis utilizing recombinant glycosyltransferases. Subsequent conjugation of these glycans to lysine groups of bovine serum albumin via squaric acid diethyl ester yielded a set of 22 different neo-glycoproteins with tuned ligand density. The neo-glycoproteins were analyzed by biochemical and chromatographic methods proving various modification degrees. The neo-glycoproteins were used for binding and inhibition studies with human galectin-3 showing high affinity. Binding strength and inhibition potency are closely related to modification density and show binding enhancement by multivalent ligand presentation. At galectin-3 concentrations comparable to serum levels of cancer patients, we detect the highest avidities. Selectivity of N,N-diacetyllactosamine terminated structures towards galectin-3 in comparison to galectin-1 is demonstrated. Moreover, we also see strong inhibitory potency of our scaffolds towards galectin-3 binding. These novel neo-glycoproteins may therefore serve as selective and strong galectin-3 ligands in cancer related biomedical research.

In Vivo Nanotoxicity Testing using the Zebrafish Embryo Assay.

Nanoparticles are increasingly used for biomedical purposes. Many different diagnostic and therapeutic applications are envisioned for nanoparticles, but there are often also serious concerns regarding their safety. Given the fact that numerous new nanomaterials are being developed every day, and that not much is known about the long-term toxicological impact of exposure to nanoparticles, there is an urgent need to establish efficient methods for nanotoxicity testing. The zebrafish (Danio rerio) embryo assay has recently emerged as an interesting 'intermediate' method for in vivo nanotoxicity screening, enabling (semi-) high-throughput analyses in a system significantly more complex than cultured cells, but at the same time also less 'invasive' and less expensive than large-scale biocompatibility studies in mice or rats. The zebrafish embryo assay is relatively well-established in the environmental sciences, but it has not yet gained wide notice in the nanomedicine field. Using prototypic polymeric drug carriers, gold-based nanodiagnostics and nanotherapeutics, and iron oxide-based nanodiagnostics, we here show that toxicity testing using zebrafish embryos is easy, efficient and informative, and faithfully reflects, yet significantly extends, cell-based toxicity testing. We therefore expect that the zebrafish embryo assay will become a popular future tool for in vivo nanotoxicity screening.