Eight pyrrolidine, five pyrrolizidine and one indolizidine analogue(s) of the known alpha-mannosidase inhibitor, the azafuranose, 1,4-dideoxy-1,4-imino-D-mannitol (DIM), have been tested for inhibition of the multiple forms of alpha-mannosidase in human liver in vitro. Substitution of the ring nitrogen markedly decreased or abolished inhibition, but loss of the C-6 hydroxy group, as in 6-deoxy-DIM and 6-deoxy-6-fluoro-DIM, enhanced inhibition, particularly of the lysosomal alpha-mannosidase. Addition of the anomeric substituent-CH2OH decreased inhibition. To be a potent inhibitor of the lysosomal, Golgi II and neutral alpha-mannosidases, a polyhydroxylated pyrrolidine must have the same substituents and chirality as mannofuranose at C-2, C-3, C-4 and C-5. These four chiral centres can also be part of a polyhydroxylated indolizidine, e.g. swainsonine, but not of a pyrrolizidine, e.g. cyclized DIM, ring-contracted swainsonine or 1,7-diepi-australine. DIM did not inhibit lysosomal alpha-mannosidase intracellularly, but both 6-deoxy-DIM and 6-deoxy-6-fluoro-DIM caused accumulation of partially catabolized glycans in normal human fibroblasts. Analysis of these induced storage products by h.p.l.c. showed that both compounds also inhibited Golgi alpha-mannosidase II and that 6-deoxy-6-fluoro-DIM was also a good inhibitor of the endoplasmic reticulum alpha-mannosidase and specific lysosomal alpha (1-6)-mannosidase. None of the mannofuranose analogues appeared to inhibit Golgi alpha-mannosidase I.
The specificity of human liver lysosomal alpha-mannosidase (EC 3.2.1.24) towards a series of oligosaccharide substrates derived from high-mannose, complex and hybrid asparagine-linked glycans and from the storage products in alpha-mannosidosis was investigated. The enzyme hydrolyses all alpha(1-2)-, alpha(1-3)- and alpha(1-6)-mannosidic linkages in these glycans without a requirement for added Zn2+, albeit at different rates. A major finding of this study is that all the substrates are hydrolysed by non-random pathways. These pathways were established by determining the structures of intermediates in the digestion mixtures by a combination of h.p.t.l.c. and h.p.l.c. before and after acetolysis. The catabolic pathway for a particular substrate appears to be determined by its structure, raising the possibility that degradation occurs by an uninterrupted sequence of steps within one active site. The structures of the digestion intermediates are compared with the published structures of the storage products in mannosidosis and of intact asparagine-linked glycans. Most but not all of the digestion intermediates derived from high-mannose glycans have structures found in intact asparagine-linked glycans of human glycoproteins or among the storage products in the urine of patients with mannosidosis. However, the relative abundances of these structures suggests that the catabolic pathway is not the same as the processing pathway. In contrast, the intermediates formed from the digestion of oligosaccharides derived from hybrid and complex N-glycans are completely different from any processing intermediates and also from the oligosaccharides of composition Man2-4GlcNAc that account for 80-90% of the storage products in alpha-mannosidosis. It is postulated that the structures of these major storage products arise from the action of an exo/endo-alpha(1-6)-mannosidase on the partially catabolized oligomannosides that accumulate in the absence of the main lysosomal alpha-mannosidase.
The synthetic amino sugar 1,4-dideoxy-1,4-imino-L-allitol (DIA) is a moderately good inhibitor of human liver alpha-D-mannosidases and a weak inhibitor of alpha-L-fucosidase, N-acetyl-beta-D-hexosaminidase and beta-D-mannosidase. Methylation of the ring nitrogen of DIA markedly decreases the inhibition of all the glycosidases except N-acetyl-beta-D-hexosaminidase. N-Benzylation of DIA essentially abolishes all inhibitory activity, except towards alpha-L-fucosidase, which is more strongly inhibited than by either DIA or N-methyl-DIA. This is the first report of a change of specificity of inhibition of a glycosidase inhibitor by substitution of the ring nitrogen.
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