Synergistic Inhibition of Human R-1,3-Fucosyltransferase V Lei Qiao, ² Brion W. Murray, ² Makoto Shimazaki, ² Jody Schultz, ‡ and Chi-Huey Wong* ,² Contribution from the Department of Chemistry, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California, 92037, and Cytel Corporation, 3525 John Hopkins Court, San Diego, California, 92121 ReceiVed January 25, 1996 X Abstract: Human R-1,3-fucosyltransferase V (FucT V), which catalyzes the transfer of L-fucose moiety from guanosine diphosphate -L-fucose (GDP-Fuc) to an acceptor sugar to form sialyl Lewis x (sLe x ), was shown to proceed through an ordered, sequential mechanism by product inhibition studies. The designed azatrisaccharide propyl 2-acetamido- 2-deoxy-4-O-(-D-galactopyranosyl)-3-O-(2-(N-(-L-homofuconojirimycinyl))ethyl)-R-D-glucopyranoside (2), prepared by covalently linking the N-group of -L-homofuconojirimycin (1) to the 3-OH of LacNAc through an ethylene unit, in the presence of GDP was found to be an effective inhibitor of FucT V. In the presence of 30 µM GDP, the concentration of 2 necessary to cause 50% inhibition was reduced 77-fold to 31 µM. Presumably, the azatrisaccharide and GDP form a complex which mimics the transition state of the enzymatic reaction. Given the low affinity of FucT V for its substrate LacNAc (K m ) 35 mM), the designed azatrisaccharide in the presence of GDP represents the most potent synergistic inhibitor complex reported so far. Introduction Many antigenic oligosaccharides on the cell surface are fucosylated. These fucose-containing structures are regarded as oncodevelopmental antigens since they accumulate in a large variety of human cancers. 1 The biosynthesis of these structures requires the action of several glycosyltransferases, of which fucosylation by a class of fucosyltransferases (FucT) is the last and critical step. 2 R-L-Fucosidase 3 is a degradative enzyme which is involved in the hydrolytic removal of fucose residue from these glycoconjugates. Studies have indicated that in- creased activities of fucosyltransferase and R-L-fucosidase are responsible for the abnormal expression of these fucose- containing antigens in endometrial carcinoma. 4 Therefore, inhibitors of Fuc-T and R-fucosidase are potentially useful as anti-inflammatory and anti-tumor agents. Fucosyltransferases catalyze the transfer of the L-fucose moiety from guanosine diphosphate -L-fucose (GDP-Fuc) to the corresponding glycoconjugate acceptors to form an R-1,2, R-1,3/4, R-1,3 or R-1,6 linkage. 5 Five human R-1,3-fucosyl- transferases have been cloned and mapped on chromosomes; 6 among them, R-1,3-fucosyltransferase V (FucT V) has been shown to be responsible for the production of sialyl Lewis x (sLe x , Figure 1), a ligand for E-selectin involved in inflammatory process and tumor development. 7 This enzyme has been studied for acceptor specificity and used in the chemoenzymatic synthesis of sLe x . 8,9 This enzyme accepts both N-acetyllac- tosamine (LacNAc) and sialyl LacNAc as the substrates with K m values of 35 and 100 mM, respectively. The related enzyme R-1,2-fucosyltransferase has been hypothesized to proceed through a simple ion-pair transition state which results in the inversion of anomeric configuration, 10 R-1,3-fucosyltransferases may operate Via a similar mechanism involving a displacement of GDP by the acceptor hydroxyl group assisted by a base on the enzyme (Figure 2). To date there is no X-ray crystal structure of any glycosyltransferase reported and none of the glycosyltransferases have been studied in detail with respect to their mechanism, though the kinetic mechanisms of a limited ² The Scripps Research Institute. ‡ Cytel Corp. X Abstract published in AdVance ACS Abstracts, August 1, 1996. (1) (a) Hakomori, S. AdV. Cancer Res. 1989, 52, 257. (b) Hakomori, S.; Nudelman, E.; Levery, S. B.; Kannagi, R. J. Biol. Chem. 1984, 259, 4672. (c) Feizi, T. Nature 1985, 314, 53. (2) (a) Natsuka, S.; Lowe, J. B. Curr. Opin. Struct. Biol. 1994, 4, 683. (b) Holme, E. H.; Ostrander, G. K.; Hakomori, S. J. Biol. 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