Abstract

In cancers, increased fucosylation of cell surface glycans is a trademark of malignant cell transformation and contributes to many aberrant cellular events during cancer development. Alterations in the expression of fucosylated oligosaccharides is mainly attributed to the upregulation of specific fucosyltransferase enzymes (FUTs), which construct fucosylated glycans using GDP-fucose as a substrate. FUT5 and FUT6 are two important enzymes of the a1-3-fucosyltransferase family that act to form sialyl LewisX, a member of the Lewis antigen family that is abnormally abundant in numerous cancers and is associated with advanced tumor grade poor prognosis due to its capacity to interact with E- and L- selectin molecules expressed by the endothelium, thus aiding in metastasis by direct binding of cancer cells.
To identify inhibitors of FUT5 and FUT6 enzymes as potential cancer therapeutics, we have developed a high-throughput coupled enzyme assay that makes use of 3’-sialyl-N-acetyllactosamine-resorufin (3’-SLN-Res), a fluorogenically labeled sialyl LewisX precursor. When non-fucosylated, this synthetic substrate is recognized and hydrolyzed by specific glycoside hydrolase enzymes (glycosidases) to release fluorescent resorufin, yet if the probe is fucosylated by FUT5 or FUT6 prior to treatment with glycosidases, these cannot act on the substrate and no fluorescent signal is produced. Employing this assay, we were able to screen a library of bioactive small-molecule compounds in a fully automated high-throughput screening and identify several hits, demonstrating that our approach can be used to single out potent FUT inhibitors from commercial compound libraries in a microtiter plate format.