Design and Development of Galectin-Specific Inhibitors

Abstract

Galectins are a family of carbohydrate binding proteins found in the human body and are involved in the modulation of vital biological functions. These biological functions can be initiated through interactions formed between the conserved carbohydrate recognition domain and β-galactoside-containing glycoconjugates. Due to their modulatory roles in cell homeostasis, galectins have been implicated in the progression of different diseases such as in tumours, cancers and inflammatory disorders, making them novel therapeutic targets. The activities of galectin can be inhibited by blocking interactions with its natural ligands and protein binding partners, through the design and development of small carbohydrate ligands. The amino acid residues that partake in the binding of carbohydrate ligands are highly conserved across the galectin family, leading to ligands binding in a very specific manner. To avoid cross-reactivity with other galectins in the body which could be detrimental in drug therapies, the development of selective inhibitors is essential. Galectin-3 and -4C have been determined to play roles in the progression of cancer and different inflammatory disorders and are the primary focus for this research project. The research studies presented and discussed in this thesis, revolve around the use of structural determination and computational techniques to identify and inform on the intricacies of the galectin binding site, to help aid in the development of galectin-specific inhibitors.\nIn order to develop a better understanding of the binding specificities of the different galectins towards particular ligands, an in silico study was conducted to identify small differences in the galectin binding site, which may influence the physical shape and properties of the binding pocket (Chapter 2). From a series of molecular dynamic simulations of wildtype and mutant galectins (galectin-1, -3, -4, -7, -8 and -9), a network of salt-bridge interactions and large bulky side chain amino acids were identified within the binding site and were found to influence the binding of ligands to specific galectins. These subtle differences between the different galectins could be exploited in future ligand design campaigns. As galectin-3 and -4 have shown similar affinities towards multiple ligand scaffolds, the further differences between the two were investigated in Chapter 3. A series of galactocoumaryl derivatives were designed and synthesized, where the 6th position of the coumarin ring (16a) was modified to include either a methyl (16b), a methoxy (16c) or a chloro group (16d). Their ligand binding conformations were determined using X-ray crystallography and affinities tested using SPR and ITC. Galectin-3 exhibited a two times stronger affinity towards all four ligands compared to galectin-4C, which is attributed to the presence of Arg144. An X-ray crystal structure of a modified C1, C3-galactose ligand, selective towards galectin-4C (provided by a collaborator, Prof. Ulf Nilsson) is also reported in Chapter 3. The structural information reported in this chapter will help fine-tune the selectivity of future inhibitors towards galectin-3 or -4C.\nHighly specific inhibitors can be further developed through the use of varying carbohydrate scaffolds. The development of talose-based inhibitors has been a novel direction within the Blanchard research group, with current developed inhibitors binding to galectin-3 and -4C in the single digit micromolar range. As no structural information is available for galectin-4C with these taloside inhibitors, a molecular dynamic simulations study was pursued. The simulations revealed that the enhanced binding affinity of novel taloside inhibitors towards galectin-4C is due to the formation of an open binding cavity that lacks arginines and other bulky side chain amino acids, allowing for strong interactions to be formed with the talose ligands (Chapter 3). In Chapter 4, an X-ray crystallography study was employed to determine the ligand binding conformations of novel disaccharide scaffolds, selenodigalactoside and diselenodigalactoside (provided by a collaborator, Prof. Laszlo Szilagyi) in galectin-3 and lactulose in galectin-4C (Chapter 4). Selenodigalactoside was determined to adopt typical disaccharide ligand conformations in galectins, offering an alternative ligand scaffold. Lactulose exhibited single digit micromolar affinity towards both domains of galectin-4, as determined through SPR. X-ray crystallography and molecular dynamic simulations revealed that the enhanced binding of galectin-4 towards lactulose is due to the formation of an additional hydrogen bond interaction between the C6′-OH atom of the distal fructose ring and a conserved glycine residue. A series of lactulose derivatives were also designed and tested in silico against galectin-3, -4C and -4N D69A, to help aid in future ligand design campaigns utilising the lactulose scaffold.\nThe use of molecular dynamic simulations combined with other techniques such as X-ray crystallography and binding affinity assays, can provide information on the protein binding site aiding in ligand development. These techniques were utilised in this thesis to develop a better understanding of the galectin binding site and to help design and develop different ligands utilising novel scaffolds. The design of highly specific inhibitors is needed to help aid in the inhibition of specific galectins and their functions in different diseased states. Overall, the research presented in this thesis, has provided information to aid in the further fine-tuning of ligand specificity towards particular galectins.