Exploration of the active site of β4GalT7 – Synthesis of substrates and inhibitors

Abstract: β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of proteoglycans (PGs) and glycosaminoglycan (GAG) chains, which are important macromolecules involved in many biological processes such as cell growth and cell signaling as well as in cancer pathobiology and viral and bacterial infections. Despite its pivotal role, much is still unknown regarding the specific structure of GAGs and how the structure affects its functions. GAG synthesis can be regulated by xylosides acting on β4GalT7 as either substrates or inhibitors. In an effort to find efficient substrates and inhibitors of β4GalT7, which would be valuable tools applicable in GAG research, we set out to investigate the active site of β4GalT7 by synthesizing and examining xylosides and xyloside analogs with modifications in the xylose moiety, the endocyclic and exocyclic positions, as well as the aglycon. The overarching aim with these investigations was to pinpoint the requirements of efficient substrates and efficient inhibitors. The synthesized compounds were evaluated in a β4GalT7 assay in combination with molecular docking simulations and conformational analysis by NMR spectroscopy. We found that efficient substrates are formed when keeping the xylose moiety unmodified, but exchanging the endocyclic and/or exocyclic oxygen atoms for sulfur, as well as connecting the xyloside to an aglycon consisting of a fused aromatic system, preferably separated from the xylose part by a short oligoethylene glycol spacer. These structural features can not directly be transferred to a xyloside analog possessing inhibitory activity to gain efficient inhibitors of β4GalT7.