Design, synthesis and evaluation of sialic acid-based mimetics as siglec-8 and galectin-8 inhibitors

Siglecs are a family of sialic acid-binding immunoglobulin (Ig)-like lectins, which are single-pass transmembrane proteins found on the surface of human cells. Siglec-8, a human immune-inhibitory receptor, is expressed on eosinophils, basophils, and mast cells. When siglec-8 binds to antibodies or glycan ligands, it triggers apoptosis in eosinophils and inhibits the release of mediators from mast cells, without affecting their stability. Consequently, glycan ligands targeting siglec-8 hold potential as inhibitors for treating eosinophil- and mast cell-related diseases, such as asthma, chronic rhinosinusitis, chronic urticaria, hypereosinophilic syndromes, as well as mast cell and eosinophil malignancies and eosinophilic gastrointestinal disorders.In a previous study, mimetics of 6’-sulfo-sLex, a specific glycan ligand for siglec-8, were identified. Keeping neuraminic acid (sialic acid), while replacing the galactopyranose with a cyclohexyl derivative led to a higher affinity ligand with significantly reduced carbohydrate character. Here, I present design and synthesis of C9-naphthylsulfonamide sialyl triazoles where the sulfated galactose residue is replaced by a triazole ring carrying a carboxylate or sulfonate. The affinity of the best representative 36 with a KD of 45.5 µM (determined by ITC) has 6-fold improved affinity compared to the natural ligand 6’-sulfo-sialyl Lewisx. In addition, the C-5 acetamide was further modified to hydroxyl methyl acetamide, resulting in compound 56. Molecular dynamics simulation (100 ns) was performed predicting several key interactions: the hydroxyl group of hydroxymethyl acetamide forms hydrogen bonds with residues Lys132 and Tyr27 and the sulfonate group engages in salt-bridge interactions with Arg72 and hydrogen bonding with Gln75 and Tyr74 of Siglec-8. Also, O-glycoside based sialyl mimetics functionalized with triazole-linked carboxylates (compounds 66 and 67) were synthesized but found to be suboptimal for binding with siglec-8. These results provide new insights for the development of more potent siglec-8 inhibitors. Galectins are carbohydrate-binding proteins that specifically bind with various β-galactosides. Galectin-8 plays a crucial role in regulating cancer growth, metastasis, tumour progression, and tumour cell survival. Notably, galectin expression is often higher in tumour tissues compared to non cancerous tissues. Galectin-8 also plays a role in defending against bacterial and viral infections, as well as in immune regulation. Galectin-8 contains two distinct carbohydrate recognition domains (CRDs, Gal8N and Gal8C), and selective inhibitors targeting at least one of these CRDs are valuable for both galectin-8 biology research and potential pharmacological applications.Gal-8N shows a preference for binding to 3’-sialyllactose with a KD of 2.3 µM, while its derivative, sialyl galactose, exhibits reduced affinity with a KD of 150 µM. Here, I have designed macrocyclic mimetics (M1-M6) based on disaccharides composed of sialic acid and galactose to constrain the flexible sialic acid-galactopyranose linkage by: i) connecting the anomeric position of the galactose moiety to the C9 position of the sialic acid moiety, and ii) connecting the C2 position of the galactose moiety to the C7 position of the sialic acid moiety. The successful synthesis of macrocycle M2 was achieved, leading to a substance exhibiting a KD of 40 μM, which represents a 4-fold improvement compared to NeuAcalpha(2-3)Galbeta1-OMe. Metadynamics studies revealed a single low-energy conformer for the macrocycles (M1, M2, M5, M6-β), which closely align with the binding conformation of the natural ligand, 3’-sialyllactose, as observed in its crystal structure complex with galectin-8N. These macrocycles provide new insights, as further functional group modifications could lead to more potent galectin-8N inhibitors.

Funder

Research Ireland

Publisher

University of Galway

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CC BY-NC-ND

cbn

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University of Galway Theses (PhD Theses)