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 -伊杜醛酸和d -氨基葡萄糖相关的gemine -二胺1- n -亚糖分别抑制Hunter综合征和Sanfilipo综合征中用于gag链生物合成的伊杜醛酸2- o -硫基转移酶（2-O-ST）和n -去乙酰化酶/ n -硫基转移酶（NDST）。在三年内，大约有60种化合物被合成，并通过体外酶分析评估为2-O-ST和NDST的抑制剂。两种含有胍基的亚氨基糖作为2-O-ST的有效抑制剂。随后在体内测试了两种抑制剂作为糖胺聚糖生物合成抑制剂，使用(i)生物素化的FGF-2 （ii）总^<35> so_4标记糖胺聚糖链和（iii） ^ 3h - glcn标记糖胺聚糖链，然后进行双糖分析。然而，没有一种体外活性化合物是体内GAG生物合成的抑制剂。这两种化合物也被发现对CHO-K1细胞无毒。缺乏活性很可能反映了由于带正电的胍部分而导致细胞摄取不良。这些生物学研究可能需要引入取代基和/或去除阻断基团来增加化合物对细胞膜的渗透性。这是第一个证据，宝石二胺1- n -亚氨基糖作为一种酶的抑制剂参与硫酸肝素合成。这表明底物剥夺疗法和酶替代疗法的结合也将是预期的。

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.