Selective modifications of lactose and N-acetyllactosamine with sulfate and aromatic bulky groups unveil unique structural insights in galectin-1-ligand recognition.

Galectins, a family of endogenous glycan-binding proteins, play crucial roles in a broad range of physiological and pathological processes. Galectin-1 (Gal-1), a proto-type member of this family, is overexpressed in several cancers and plays critical roles in tumor-immune escape, angiogenesis and metastasis. Thus, generation of high-affinity Gal-1 inhibitors emerges as an attractive therapeutic approach for a wide range of neoplastic conditions. Small-molecule carbohydrate inhibitors based on lactose (Lac) and N-acetyllactosamine (LacNAc) structures have been tested showing different results. In this study, we evaluated Lac- and LacNAc-based compounds with specific chemical modifications at key positions as Gal-1 ligands by competitive solid-phase assays (SPA) and isothermal titration calorimetry (ITC). Both assays showed excellent correlation, highlighting that lactosides bearing bulky aromatic groups at the anomeric carbon and sulfate groups at the O3' position exhibited the highest binding affinities. To dissect the atomistic determinants for preferential affinity of the different tested Gal-1 ligands, molecular docking simulations were conducted and PRODIGY-LIG structure-based method was employed to predict binding affinity in protein-ligand complexes. Notably, calculated binding free energies derived from the molecular docking were in accordance with experimental values determined by SPA and ITC, showing excellent correlation between theoretical and experimental approaches. Moreover, this analysis showed that 3'-O-sulfate groups interact with residues of the Gal-1 subsite B, mainly with Asn33, while the ester groups of the aromatic anomeric group interact with Gly69 and Thr70 at Gal-1 subsite E, extending deeper into the pocket, which could account for the enhanced binding affinity. This study contributes to the rational design of highly optimized Gal-1 inhibitors to be further studied in cancer models and other pathologic conditions.

Selectively Modified Lactose and N-Acetyllactosamine Analogs at Three Key Positions to Afford Effective Galectin-3 Ligands.

Galectins constitute a family of galactose-binding lectins overly expressed in the tumor microenvironment as well as in innate and adaptive immune cells, in inflammatory diseases. Lactose ((ß-D-galactopyranosyl)-(1→4)-ß-D-glucopyranose, Lac) and N-Acetyllactosamine (2-acetamido-2-deoxy-4-O-ß-D-galactopyranosyl-D-glucopyranose, LacNAc) have been widely exploited as ligands for a wide range of galectins, sometimes with modest selectivity. Even though several chemical modifications at single positions of the sugar rings have been applied to these ligands, very few examples combined the simultaneous modifications at key positions known to increase both affinity and selectivity. We report herein combined modifications at the anomeric position, C-2, and O-3' of each of the two sugars, resulting in a 3'-O-sulfated LacNAc analog having a Kd of 14.7 µM against human Gal-3 as measured by isothermal titration calorimetry (ITC). This represents a six-fold increase in affinity when compared to methyl ß-D-lactoside having a Kd of 91 µM. The three best compounds contained sulfate groups at the O-3' position of the galactoside moieties, which were perfectly in line with the observed highly cationic character of the human Gal-3 binding site shown by the co-crystal of one of the best candidates of the LacNAc series.

Synthesis & Evaluation of Novel Mannosylated Neoglycolipids for Liposomal Delivery System Applications.

Glycosylated NPs, including liposomes, are known to target various receptors involved in cellular carbohydrate transport, of which the mannoside binding receptors are attracting particular attention for their expression on various immune cells, cancers, and cells involved in maintaining central nervous system (CNS) integrity. As part of our interest in NP drug delivery, mannosylated glycoliposomal delivery systems formed from the self-assembly of amphiphilic neoglycolipids were developed, with a C12-alkyl mannopyranoside (ML-C12) being identified as a lead compoundcapable of entrapping, protecting, and improving the delivery of structurally diverse payloads. However, ML-C12 was not without limitations in both the synthesis of the glycolipids, and the physicochemical properties of the resulting glycoliposomes. Herein, the chemical syntheses of a novel series of mannosylated neoglycolipids are reported with the goal of further improving on the previous ML-C12 glyconanoparticles. The current work aimed to use a self-contingent strategy which overcomes previous synthetic limitations to produce neoglycolipids that have one exposed mannose residue, an aromatic scaffold, and two lipid tails with varied alkyl chains. The azido-ending carbohydrates and the carboxylic acid-ending lipid tails were ligated using a new one-pot modified Staudinger chemistry that differed advantageously to previous syntheses. The formation of stable neoglycoliposomes of controllable and ideal sizes (≈100-400 nm) was confirmed via dynamic light scattering (DLS) experiments and transmission electron microscopy (TEM). Beyond chemical advantages, the present study further aimed to establish potential improvements in the biological activity of the neoglycoliposomes. Concanavalin A (Con A) agglutination studies demonstrated efficient and stable cross-linking abilities dependent on the length of the linkers and lipid tails. The efficacy of the glycoliposomes in improving cytosolic uptake was investigated using Nile Red as probe in immune and cancer cell lines. Preliminary ex vivo safety assessments showed that the mannosylated glycoliposomes are hemocompatible, and non-immunogenic. Finally, using a model peptide therapeutic, the relative entrapment capacity and plasma stability of the optimal glycoliposome delivery system was evaluated and compared to the previous neoglycoliposomes. Overall, the new lead glycoliposome showed improved biological activity over ML-C12, in addition to having several chemical benefits including the lack of stereocenters, a longer linker allowing better sugar availability, and ease of synthesis using novel one-pot modified Staudinger chemistry.

Practical non-enzymatic synthesis of propargyl sialyl-α-(2-3')-lactosamine trisaccharide using minimal protecting groups manipulation.

The trisaccharide, prop-2-ynyl 5-acetamido-3,5-dideoxy-d-glycero-α-d-galacto-2-nonulopyranosylonic acid-(2 â†’ 3)-ß-d-galactopyranosyl-(1 â†’ 4)-2-acetamido-2-deoxy-ß-d-glucopyranoside (9) has been efficiently synthesized in a few steps without the need of conformationally constrained glycosyl donors and acceptors or enzymes. First, using the known prop-2-ynyl 2-acetamido-2-deoxy-6-O-tert-butyldiphenylsilyl-ß-d-glucopyranoside as acceptor (2) and the peracetylated galactosyl trichloroacetimidate (3) as glycosyl donor, followed by protecting groups manipulation, prop-2-ynyl (6-O-tert-butyldiphenylsilyl-ß-d-galactopyranosyl)-(1 â†’ 4)-2-acetamido-2-deoxy-6-O-tert-butyldiphenylsilyl-ß-d-glucopyranoside (6) was synthesized with exclusive O-4 regioselectivity due to steric hindrance of the upper face of the acceptor at O-3. Sialylation with the thiophenyl glycosyl donor (7) afforded the desired trisaccharide with the shortest number of steps and in higher overall yield than previously reported methodologies. The direct use of minimally protected N-acetyl-lactosamine acceptor (6) was critical for the efficient synthesis of the title compound. The propargylic aglycone is suitable for chemical ligation using click chemistry as reported for its (2 â†’ 6) sialylated analog.

Recent Development in the Design of Neoglycoliposomes Bearing Arborescent Architectures.

This brief review highlights systematic progress in the design of synthetic glycolipid (neoglycolipids) analogs evolving from the conventional architectures of natural glycosphingolipids and gangliosides. Given that naturally occurring glycolipids are composed of only one hydrophilic sugar head-group and two hydrophobic lipid tails embedded in the lipid bilayers of the cell membranes, they usually require extraneous lipids (phosphatidylcholine, cholesterol) to confer their stability. In order to obviate the necessity for these additional stabilizing ingredients, recent investigations have merged dendrimer chemistry with that of neoglycolipid syntheses. This singular approach has provided novel glycoarchitectures allowing reconsidering the necessity for the traditional one to two hydrophilic/hydrophobic ratio. An emphasis has been provided in the recent design of modular arborescent neoglycolipid syntheses coined glycodendrimersomes.

Development of LM98, a Small-Molecule TEAD Inhibitor Derived from Flufenamic Acid.

The YAP-TEAD transcriptional complex is responsible for the expression of genes that regulate cancer cell growth and proliferation. Dysregulation of the Hippo pathway due to overexpression of TEAD has been reported in a wide range of cancers. Inhibition of TEAD represses the expression of associated genes, demonstrating the value of this transcription factor for the development of novel anti-cancer therapies. We report herein the design, synthesis and biological evaluation of LM98, a flufenamic acid analogue. LM98 shows strong affinity to TEAD, inhibits its autopalmitoylation and reduces the YAP-TEAD transcriptional activity. Binding of LM98 to TEAD was supported by 19 F-NMR studies while co-crystallization experiments confirmed that LM98 is anchored within the palmitic acid pocket of TEAD. LM98 reduces the expression of CTGF and Cyr61, inhibits MDA-MB-231 breast cancer cell migration and arrests cell cycling in the S phase during cell division.

Influence of protecting groups on O- and C-glycosylation with neuraminyl and ulosonyl dibutylphosphates.

The adamantanyl thioglycosides of 5-isothiocyano and 5-azido 5-desamino-4,7,8,9-tetra-O-acetylneuraminic acid methyl ester were converted into the corresponding dibutyl phosphates, which proved to be excellent α-selective donors for O-sialidation with a range of typical acceptors, and good donors for reaction with allyltributylstannane, albeit without significant anomeric selectivity. In the KDN series the dibuylphosphate derived from a donor carrying a 4,5-cyclic carbonate protecting group afforded the corresponding C-glycoside with excellent α-selectivity on activation in the presence of allyltributylstannane, whereas the corresponding donor carrying acetate esters at the 4- and 5-positions was unselective. Overall, it is revealed that while the strongly electron-withdrawing isothiocyanato and azido groups are sufficient to promote highly α-selective O-sialidation, they are inadequate when faced with less reactive nucleophiles when mixtures of anomers are obtained.

Tetraphenylethylene-based glycoclusters with aggregation-induced emission (AIE) properties as high-affinity ligands of bacterial lectins.

Tetraphenylethylene (TPE) is fluorescent through aggregation induced emission (AIE) in water. Herein, TPE was used as the core of glycoclusters that target the bacterial lectins LecA and LecB of Pseudomonas aeruginosa. Synthesis of these TPE-based glycoclusters was accomplished by using azide-alkyne "click" chemistry. The AIE properties of the resulting glycoclusters could be readily verified, but imaging could not be pursued due to the overlap of the fluorescence signals from cells and bacteria. Nonetheless, the glycoclusters displayed nanomolar affinities toward LecA and LecB. Further evaluation in a cell-based anti-adhesive assay highlighted a limited decrease in adhesion (20%) for the fucosylated glycocluster. This confirmed that these TPE-based glycoclusters are indeed LecA and LecB high-affinity ligands. Nevertheless, the hypotheses involving their application in imaging or anti-adhesive therapy could not be verified.