Structure based design of inhibitors targeting galectin-8

Abstract

Galectins are β-galactoside binding proteins that are found in all type of living organisms, and involved in many physiological functions such as inflammation, immune responses, cell adhesion, growth and migration, apoptosis, etc. Due to their association with the progression of several metabolic and disease conditions, galectins are recognised as important targets for the drug development. Galectin-8 is involved in several biological functions such as cell adhesion and growth, immune responses, inflammation, new blood vessel formation, osteoblast and osteoclast differentiation, cancer growth and metastasis, platelet aggregation. Structurally, galectin-8 is a tandem-repeat type galectin, comprising an N-terminal and (galectin-8N) C-terminal (galectin-8C) CRD domain connected by a peptide linker. Superimposition of both the CRDs reveals structural differences between their carbohydrate binding sites. The individual N- and C-CRD domain shows similar functional roles which are observed with full length galectin-8, however, their potency is less compared to full length galectin-8. Several structural investigations showed that the N-terminal domain preferentially binds to the anionic sialylated, sulphated oligosaccharides due to the presence of the unique Arg59, which is only present in galectin-8N and hence is unique amongst all galectins. The main body of research presented in this thesis is about design, synthesis of selective and potent galectin-8N antagonists by utilising a structure-based drug design approach. Our previous structural investigation revealed unique amino acids of the galectin-8N binding site such as Arg59, Gln47, Tyr141. The structure-based ligand design campaign was further continued by targeting these unique amino acids of the N-terminal carbohydrate binding site. The novel compounds have been successfully designed and chemically synthesized. Galectin-8 protein expression (in E. coli) and affinity-based purification (using Lactosyl-Sepharose column) were performed to obtain the purified protein. The binding affinity of the synthesized compounds against galectin-8N were evaluated by ITC. X-ray crystallography was carried out to determine the binding interactions of compounds with the galectin-8N carbohydrate binding site. These compounds were further evaluated in cell culture studies to determine their ability to inhibit cancer promoting gene expression during my visit to our collaborator Prof. Yehiel Zick’s research laboratory, at the Weizmann Institute of Science, Israel. It was reported that disaccharide lactose has stronger binding affinity than the monosaccharide galactose due to significantly more interactions with the galectin-8N binding site. However, we chose to work with monosaccharide galactose as a pharmacophore at the beginning of the drug design campaign. For the design of galactose 3-OH substitution, the initial thought was to design an inhibitor which can cross-link two arginines (Arg45, Arg59) that are located across the binding site and thus holding the inhibitor from both sides. The primary aim was to design unique scaffolds which can cross link these arginines. After deciding the scaffold, we successfully designed, synthesized a library of galactomalonic acid derivatives and determined their in vitro binding affinity against galectin-8N. This structure-based ligand design approach led to the development of a monosaccharide based compound 42 which shows almost 7 times stronger binding affinity than the disaccharide lactose. The X-ray crystallographic structural investigation of the galectin-8N-compound 45 complex revealed the binding interactions which are responsible for the strong binding affinity of these compounds. Considering these novel research findings, the galactose-coumarin ester complexes were subsequently designed, synthesized and analysed to determine their in vitro binding affinity. The binding affinity results demonstrated that the galactose-coumarin ester complex 70 shows 5 times stronger binding affinity than disaccharide lactose. Compound 70 was further analysed in SUM159 cell culture and a mice osteoblast study. The cell culture results demonstrated that compound 70 could be a potential candidate for anti-cancer drug discovery. Our structure-based drug design campaign was further continued to design even more stronger binding affinity compound. Bis(methyl-β-D-galactopyranoside)-3-O-malonate was designed, synthesized and analysed for determining its in vitro binding affinity. The binding affinity results demonstrated that it shows almost 6 times stronger binding affinity than disaccharide lactose. Our collaborator Prof. Yehiel Zick and colleagues recently reported that galectin-8 upregulates proinflammatory cytokine and chemokine gene expression in several cell types including mice osteoblast. It was also observed that these cytokine and chemokine promotes the migration of cancer cells toward cells expressing this lectin. Based on these results, the synthesized novel compounds were analysed in cell culture and osteoblast studies during my research visit to Prof. Yehiel Zick’s research laboratory, at the Weizmann Institute of Science, Israel. The cell culture results demonstrated that the biological inhibition of these compounds follows the order of in vitro binding affinity of these compounds. Overall, the research presented in this thesis, demonstrates the successful rational medicinal chemistry application to design and development of potent galectin-8N antagonists to tackle galectin-8 associated diseases.