Deoxygenated Disaccharide Analogs as Specific Inhibitors of β1–4-Galactosyltransferase 1 and Selectin-mediated Tumor Metastasis

Abstract

The disaccharide peracetylated GlcNAc␤1-3Gal␤-O-naphthalenemethanol (disaccharide 1) diminishes the formation of the glycan sialyl Lewis X (Neu5Ac␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc; sLe X ) in tumor cells. Previous studies showed that the mechanism of action of disaccharide 1 involves three steps: (i) deacetylation by carboxyesterases, (ii) action as a biosynthetic intermediate for downstream enzymes involved in sLe X assembly, and (iii) generation of several glycans related to sLe X . In this report, we show that GlcNAc␤1-3Gal␤-O-naphthalenemethanol binds to the acceptor site of human ␤1-4-galactosyltransferase much like the acceptor trisaccharide, GlcNAc␤1-2Man␤1-6Man, which is present on N-linked glycans. The 4-deoxy analog, in which the acceptor hydroxyl group was replaced by -H, did not act as a substrate but instead acted as a competitive inhibitor of the enzyme. The acetylated form of this compound inhibited sLe X formation in U937 monocytic leukemia cells, suggesting that it had inhibitory activity in vivo as well. A series of synthetic acetylated analogs of 1 containing -H, -F, -N 3 , -NH 2 , or -OCH 3 instead of the hydroxyl groups at C-3-and C-4-positions of the terminal N-acetylglucosamine residue also blocked sLe X formation in cells. The reduction of sLe X by the 4-deoxy analog also diminished experimental tumor metastasis by Lewis lung carcinoma in vivo. These data suggest that nonsubstrate disaccharides have therapeutic potential through their ability to bind to glycosyltransferases in vivo and to alter glycan-dependent pathologic processes. . Although many of these analogs are effective in vitro, they generally do not exhibit inhibitory activity in cells due to poor membrane permeability. The large number of polar hydroxyl groups and the lack of membrane transporters for oligosaccharides in most cells presumably prevent their uptake (6).In contrast to many of the inhibitors described above, peracetylated disaccharides (e.g. acetylated Gal␤1-4GlcNAc␤-Onaphthalenemethanol (NM), acetylated Gal␤1-3GalNAc␣-O-NM, and acetylated GlcNAc␤1-3Gal␤-O-NM) inhibit sLe X biosynthesis in cells (6 -9). These compounds are taken up by cells by passive diffusion and acted on by cytoplasmic or membrane-associated carboxyesterases, which remove the acetyl groups. The compounds gain access to the biosynthetic enzymes located in the Golgi complex, where they serve as substrates, priming oligosaccharide synthesis and generating products related to O-GalNAc-linked mucin oligosaccharides. Priming in this manner diverts the assembly of the O-linked chains from endogenous glycoproteins, resulting in inhibition of expression of terminal Lewis antigens that are recognized by * This work was supported, in whole or in part, by National Institutes of Health Grants CA46462 and CA112278 (to J. D. E.) and by the Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research. The crystallographic part of this project has been funded, in whole or in part, with federal funds from NCI, National Institutes of...

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