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We have obtained the X-ray crystal structure of the galectin-8 N-terminal domain (galectin-8N) with a previously reported quinoline-galactoside ligand at a resolution of 1.6Å. Based on this X-ray structure, a collection of galactosides derivatised at O3 with triazole, benzimidazole, benzothiazole, and benzoxazole moieties were designed and synthesised. This led to the discovery of a 3-O-(N-methylbenzimidazolylmethyl)-galactoside with a Kd of 1.8%[micro]M for galectin-8N, the most potent selective synthetic galectin-8N ligand to date. Molecular dynamics simulations showed that benzimidazole-galactoside derivatives bind the non-conserved amino acid Gln47, accounting for the higher selectivity for galectin-8N. Galectin-8 is a carbohydrate-binding protein that plays a key role in pathological lymphangiogenesis, modulation of the immune system, and autophagy. Thus, the benzimidazole-derivatised galactosides represent promising compounds for studies of the pathological implications of galectin-8, as well as a starting point for the development of anti-tumour and anti-inflammatory therapeutics targeting galectin-8.

Galektini so skupina proteinov s podobnimi vezavnimi mesti za prepoznavanje ogljikovih hidratov. Osnovni skelet njihovih fizioloških ligandov pogosto vključuje sladkor galaktozo. Kljub svoji navidezni podobnosti imajo zelo raznolike fiziološke in patofiziološke vloge. Galektina-1 in -8 se na različne načine vpletata v patofiziologijo rakavih obolenj, kroničnih vnetnih bolezni ter bakterijskih in virusnih infekcij. Ker učinkovitih selektivnih zaviralcev galektinov v terapiji še ni, smo se odločili za pripravo novih sinteznih ligandov teh relativno neraziskanih proteinov. Na podlagi dostopnih informacij o vezavi in znanih kristalnih strukturah smo načrtovali tri knjižnice spojin A, B in C. Pri knjižnicah A in C smo se osredotočili predvsem na proučevanje odnosa med strukturo ligandov in njihovim delovanjem (SAR) na N-končni domeni galektina-8 (galektin-8N). S knjižnico B pa smo poleg vpogleda v SAR izvedli tudi sintezo selektivnih zaviralcev galektina-1. Na osnovi spojine 6a smo ugotovili, da z meta substitucijo benzenovega obroča, vezanega na mesto 1 triazolnega obroča, dosežemo največjo afiniteto do galektina-8N. Vezavno afiniteto smo s spojinama 39 in 42 uspeli še dodatno povečati. Z največjo afiniteto se tako na galektin-8N kot na galektin-1 veže spojina 42 (Kd gal-8N = 157 µM Kd gal-1 = 13,8 µM), ki izkazuje približno 10-kratno selektivnost za galektin-1. Pri vrednotenju ligandov iz knjižnice B smo pokazali, da sta najselektivnejši med njimi spojini 31b in 31i. Konstanta disociacije spojine 31i na galektinu-1 znaša 19,6 µM, na galektinu-3 pa zgolj 1405 µM. Spojina ima torej približno 72-krat večjo afiniteto do galektina-1 v primerjavi z galektinom-3. Prav selektivnost je zaradi velike podobnosti med vezavnimi mesti teh galektinov težko dosegljiva in predstavlja enega večjih izzivov pri načrtovanju selektivnih ligandov. S spojinami 31i, 31b, 31h, 31g in 42 smo pripravili nekatere izmed najselektivnejših, do sedaj opisanih, ligandov galektina-1. Poleg tega smo z razredom spojin B dodatno razjasnili SAR spojin s podobno strukturno formulo. Spreminjanje velikosti in lastnosti substituentov na položaju orto benzenovega obroča ter uvajanje dodatnih substituentov ne povečuje afinitete ter hkrati zmanjšuje selektivnost ligandov za galektin-1 in zato ni pravi pristop. Vezavno konformacijo ligandov smo nazadnje ovrednotili še z molekulskim sidranjem v vezavni mesti galektinov-1 in -8. Na ta način smo lahko natančneje razložili rezultate, dobljene s testiranjem afinitete. Rezultati magistrske naloge tako pomembno prispevajo k razumevanju strukturnih zahtev za doseganje selektivnosti in afinitete med strukturno podobnimi galektini. 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.