Galectins are a group of proteins with similar binding sites for carbohydrate recognition. The basic scaffold of their physiological ligands often includes the sugar galactose.
Despite their apparent similarity, they have very diverse physiological and pathophysiological roles. Galectins-1 and -8 are involved in various pathways in the pathophysiology of cancer, chronic inflammatory diseases, and bacterial and viral infections. As there are no effective selective galectin ligands in therapy yet, we decided to prepare new synthetic ligands of these relatively unexplored proteins.
Based on available information on binding and known crystal structures, we designed three libraries (A, B, and C) of compounds. For libraries A and C, we focused primarily on studying the relationship between ligand structure and their function (SAR) at the N-terminal domain of galectin-8 (galectin-8N). In addition to the SAR insight, we also performed the synthesis of selective galectin-1 ligands included in the library B.
Based on compound 6a, it was found that by meta substitution of the benzene ring attached to position 1 of the triazole ring, the highest affinity for galectin-8N is achieved. We were able to further increase the binding affinity with compounds 39 and 42. Compound 42 (Kd gal-8N = 157 µM; Kd gal-1 = 13,8 µM), which exhibits approximately 10-fold selectivity for galectin-1, binds to both galectin-8N and galectin-1 with the highest affinity.
In the evaluation of ligands from library B, we showed that the most selective among them are compounds 31b and 31i. The dissociation constant of compound 31i on galectin-1 is 19,6 µM and on galectin-3 1405 µM. The compound therefore has about 72-fold greater affinity for galectin-1 compared to galectin-3. Due to the high similarity between the binding sites of these two galectins, selectivity is difficult to achieve and represents one of the major challenges in the search for selective ligands. With ligands 31i, 31b, 31h, 31g and 42, we prepared one of the most selective galectin-1 ligands described so far. In addition, the SAR of compounds with a similar structural formula was further elucidated with compound class B. Changing the size and properties of the substituents at the ortho position of the benzene ring and introducing additional substituents does not increase the affinity and at the same time reduces the selectivity of ligands for galectin-1 and is therefore not the appropriate approach.
The binding conformation of the ligands was also evaluated by molecular docking in the binding sites of galectins-1 and -8. In this way, we were able to explain in more detail the results obtained by affinity testing. The results of the master's thesis thus make an important contribution to the understanding of the structural requirements for achieving selectivity and affinity among structurally similar galectins.
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