Selective inhibition and detection of cancer-linked galectins by carbohydrate derivatives and conjugates

Abstract

Galectins are a family of lectins that bind β-galactosides and they are involved in various processes related to cancer, e.g. apoptosis, angiogenesis and cell adhesion. The precise modes of action of these lectins are, however, still not fully understood. The ever growing number of studies concerning galectins results in a clearer picture of the functions of the proteins, however, a lot of questions remain due to conflicting data arising from the use of different research techniques. The use of specific synthetic inhibitors for galectins would be useful tools to investigate the biological mechanisms of these proteins. In addition, a functional detection assay for these lectins could also greatly contribute to the knowledge of galectins. Moreover, such an assay could also serve as a prognostic or diagnostic tool in the clinic. In this thesis we describe the synthesis of specific galectin inhibitors and the development of detection method for ligand-binding proteins implemented for galectins. The detection method was also extended to the bacterial virulence factors called verotoxins that bind the carbohydrate galabiose. In chapter 1 we discuss the galectin protein family, their structure and their involvement in biological activities and pathology. Furthermore, we highlight the efforts that have been made towards the synthesis of galectin inhibitors and the methodologies that have been developed for the detection of the lectins. In addition, the lectin shiga-like toxin and its detection are briefly discussed. Chapter 2 describes the synthesis of galectin inhibitors based on thiodigalactoside and lactosamine. The inhibitors are decorated with different aromatic groups to intensify the affinity through an arginine-arena interaction in the CRDs of galectins. The best inhibitors based on thiodigalactoside with a Kd of 13-22 nM. However, there was no selectivity between the galectins. The introduction of bulkier groupl reduced the affinity to 360 nM, which is still 120 fold better than its parent compound thiodigalactoside. The selectivity for galectin-3 over galectin-1, however, was increased to 230 fold. Similar effects were obtained with the synthesis of 4-(4-phenoxybenzyl) ether-linked lactosamine. The affinity results were compared to molecular modeling data. Inspired by the best inhibitors synthesized in chapter 1 we made three DOTA conjugated inhibitors for radiolabeling experiments. The design and synthesis of these compounds based on thiodigalactosides is described in Chapter 3. Additionally, we report the first biological data derived from radiolabeling studies with the aforementioned inhibitors. Chapter 4 describes the development of a detection method for ligand-binding proteins based on antibody capture and subsequent visualization with a fluorescent ligand. The method was originally designed for galectins. Firstly, the synthesis of a general ligand based on lactose and LacNAc is explained and then the development of the detection assay using the general fluorescent ligand. Chapter 5 elaborates on the detection method described in chapter 4 and extends the findings to a verotoxin detection method. The design and synthesis of a new ligand based on galabiose is explained and the subsequent development of a detection method for verotoxin is described.