Structure‐Based Design of a Monosaccharide Ligand Targeting Galectin‐8

Abstract

Galectin-8 is a β-galactoside-recognising protein that has a role in the regulation of bone remodelling and is an emerging new target for tackling diseases with associated bone loss. We have designed and synthesised methyl 3-O-[1-carboxyethyl]-β-d-galactopyranoside (compound 6) as a ligand to target the N-terminal domain of galectin-8 (galectin-8N). Our design involved molecular dynamics (MD) simulations that predicted 6 to mimic the interactions made by the galactose ring as well as the carboxylic acid group of 3'-O-sialylated lactose (3'-SiaLac), with galectin-8N. Isothermal titration calorimetry (ITC) determined that the binding affinity of galectin-8N for 6 was 32.8 μm, whereas no significant affinity was detected for the C-terminal domain of galectin-8 (galectin-8C). The crystal structure of the galectin-8N-6 complex validated the predicted binding conformation and revealed the exact protein-ligand interactions that involve evolutionarily conserved amino acids of galectin and also those unique to galectin-8N for recognition. Overall, we have initiated and demonstrated a rational ligand design campaign to develop a monosaccharide-based scaffold as a binder of galectin-8.

Selective Monovalent Galectin‐8 Ligands Based on 3‐Lactoylgalactoside

“…Starting from the lead compound 3‐ O ‐[1‐carboxyethyl]‐β‐ d ‐galactopyranoside ( 1 ), [24] we set out to derivatize the methyl group of the lactic acid side chain with cyclohexane and phenyl moieties; we focused on the S ‐configuration as with the R ‐configuration the added group would point away from the protein into solution (see Figure 1B). In addition, to determine the importance of the free carboxylic group for binding, we replaced it with an ester moiety (Figure 2A); a benzyl ester was chosen in order to gain possible hydrophobic contacts with the proximal Tyr141 in the binding site (Figure 2C).…”

“…The investigation of the main interactions between this ligand and the protein revealed 3‐lactoylgalactoside 1 as the minimal binding epitope, which was recently crystallized, in its racemic form, in complex with galectin‐8N. It consists of a free galactose bearing a lactate residue attached by an ether linkage in position 3 (Figure 1B) [24] …”

“…In particular, the carboxylic acid in 1 interacts with Arg59 and Gln47 forming a salt bridge and an H‐bond; moreover, a complex network of H‐bonds involves the galactose and Arg45, His65, Asn67, Asn79 and Glu89 (Figure 1). [24] Starting from this compound, we aimed to further explore the chemical space around it, in particular via ( i ) modification of the lactic acid moiety and ( ii ) by introducing an α‐thiophenyl aglycone at position 1 of d ‐galactose. These modifications improved the affinity for galectin‐8N by 200‐fold compared to the affinity of compound 1 .…”

Galectin-8, cytokines, and the storm

“…Similarly, Gal-8 direct binding at the SARS-CoV-2 coronavirus RBD, might impede viral infection. Prototypes of Gal-8 inhibitors [ 146 , 147 ] are already available. Yet, further studies are required to unravel the role of Gal-8 in tumor growth and in the immunopathogenesis of COVID-19, before considering it as a potential therapeutic target.…”

Rational Design and Synthesis of Methyl-β-d-galactomalonyl Phenyl Esters as Potent Galectin-8N Antagonists

“…This galactose-based compound showed 32 μM binding affinity with galectin-8N and no significant binding affinity with galectin-8C. 19 In this paper, we continue our drug design campaign and report the design and synthesis of galactose-based methyl-β-D-galactomalonyl phenyl esters, their binding affinity with the galectin-8N CRD, and their inhibition of galectin-8-mediated chemokines and proinflammatory cytokines expression in SUM159 breast cancer cells. Finally, protein X-ray crystallography was used to provide a structural basis for the mode of inhibition of galactomalonic acid derivatives at the CRD of galectin-8N.…”