Towards the development of fucosyltransferase 8 inhibitors

Abstract

Glycosyltransferases (GTs) biosynthesise glycoconjugates and thus play a central role in regulating the abundance and biological functions of these diverse biomacromolecules. This is most evident in cancer cells, where changes in the expression level of GTs result in the production of aberrant glycoforms. This is a hallmark of cancer. Changes in GT expression levels within cancer cells can provide them with a survival advantage, both by promoting cellular migration and by aiding in evasion of host immune responses. As such, GTs represent important emerging therapeutic targets in cancer research. The research presented in this thesis focuses on a select family of GT enzymes called fucosyltransferases (FUTs). There are 13 human FUTs and each one catalyses the formation of a glycosidic bond between fucose and a glycoconjugate. FUT8 is unique amongst the FUTs because it is the only enzyme that catalyses the installation of -1,6-L-fucopyranose on the asparagine-linked N-acetylglucosamine of N-glycans. This type of glycosylation is referred to as core fucosylation.

Core fucosylation is an emerging target in the context of cancer treatment for several reasons. Firstly, removing core fucose from antibodies enhances their ability to mediate tumour cell destruction through antibody-dependent cellular cytotoxicity (ADCC). This is because antibodies without core fucose have superior affinity for the FcγRIIIa receptor on natural killer cells. Core fucosylation has also been suggested to play an important role in tumour metastasis. FUT8 activity on the glycoprotein L1CAM in tumour cells stabilises this cell adhesion molecule and by doing so promotes cancer cell motility. Blocking FUT8 in primary melanoma tumours greatly diminishes their ability to metastasise. Finally, FUT8 has also been found to play a role in stabilising PD-1, a check point protein that negatively regulates the activity of cytotoxic T cells and the target of the blockbuster immunooncology drugs Opdivo® (Nivolumab) and Keytruda® (Pembrolizumab). Blocking FUT8 activity reduces PD-1 expression on cytotoxic T cells and enhances their ability to kill tumour cells. Collectively, these findings suggest that FUT8 inhibitors may be useful for treating cancer through tumour-intrinsic and extrinsic mechanisms, though a lot of fundamental research is still required to understand the pleiotropy of FUT8. A selective drug-like FUT8 inhibitor would go a long way to aiding in the exploration of core fucose biology.

To this end, this thesis outlines the approaches I have taken to develop FUT8 inhibitors. To begin with, a new fucose mimic, 6,6,6-trifluoro-L-fucose, was synthesised and evaluated for its ability to inhibit the biosynthesis of GDP-fucose and effectively shut down all cellular fucosylation. While this molecule does not selectively inhibit FUT8, it could be a valuable tool in the production of antibodies lacking core fucose, both in industry and academia. I have also taken a rational design approach to the development of FUT inhibitors. Compounds mimicking the GDP-fucose substrate with isosteric replacements for the pyrophosphate moiety were synthesised and evaluated for their ability to bind recombinant FUT8 in an SPR binding assay. Lastly, I have pioneered the development of a new time-resolved FRET assay for FUT enzymes to enable a high throughput screen of a lead-like molecule library.

The final chapter of this thesis departs from the topic of FUT8 to explore other types of fucosylation. In the final chapter I report the synthesis of a simple fucose-containing glycan and our attempts to generate antibodies to this antigen to facilitate further research on the Protein O-fucosyltransferase (POFUT) 2 enzyme.