Studies on synthesis of inhibitors of sulfotransferases in mucopolysaccharide biosynthesis and application of the inhibitors to substrate deprivation therapy for mucopolysaccharidoses

研究了与L-艾杜糖醛酸和D-氨基葡萄糖相关的GEM-二胺-1-N-亚胺糖分别抑制Hunter综合征和Sanfilipo综合征患者Gag链生物合成的艾杜酸2-O-磺基转移酶(2-O-ST)和N-脱乙酰酶/N-磺基转移酶(NDST)。在三年的时间里，合成了约60个化合物，并通过体外酶试验对其作为2-O-ST和NDST的抑制剂进行了评价。两个含胍基团的亚氨糖是2-O-ST的有效抑制剂。随后，通过(I)生物素化的成纤维细胞生长因子-2(Ii)总糖胺聚糖链的硫酸标记和(Iii)糖胺聚糖链的~3H-GlcN标记和双糖分析，两种抑制剂作为糖胺多糖生物合成的抑制剂在体内被测试。然而，所有的体外活性化合物都不是体内GAG生物合成的抑制剂。这两种化合物对CHO-K1细胞也没有毒性。活性的缺乏很可能反映了由于带正电的胍部分而被细胞摄取得很差。这些生物学研究可能需要引入取代基和/或去除封闭基团来增加化合物对细胞膜的通透性。这是第一个证据表明吉姆二胺1-N-亚氨糖作为参与硫酸乙酰肝素合成的酶的抑制剂。这表明底物剥夺疗法和酶替代疗法的结合也是可以预见的。

gem-Diamine 1-N-iminosugars related to L-iduronic acid and D-glucosamine were investigated to inhibit iduronate 2-O-sulfotransferase (2-O-ST) and N-deacetylase/N-sulfotransferase (NDST) for the GAG-chain biosynthesis in Hunter and Sanfilipo syndrome, respectively. Within the three years, about 60 compounds were synthesized and evaluated as inhibitors of 2-O-ST and NDST using an in vitro enzyme assay. Two iminosugars containing guanidine groups acted as potent inhibitors of 2-O-ST. Two inhibitors were subsequently tested in vivo as inhibitors of glycosaminoglycan biosynthesis using (i) biotinylated FGF-2 (ii) total ^<35>SO_4-labeling of the glycosaminoglycan chains and (iii) ^3H-GlcN-labeling of the glycosaminoglycan chains followed by disaccharide analysis. However, none of the active in vitro compounds were inhibitors of GAG biosynthesis in vivo. Both compounds were also found non-toxic to CHO-K1 cells. The lack of activity most likely reflects poor uptake by cells due to the positively charged guanidine moiety. These biological studies may require introduction of substituents and/or removal blocking groups to increase the permeability of the compounds to cell membranes.This is the first evidence that gem-diamine 1-N-iminosugars act as inhibitors of an enzyme involved in heparan sulfate synthesis. This suggests that the combination of substrate deprivation therapy and enzyme replacement therapy would also be anticipated.