Module Assembly and Evaluation of Artificial Glycosaminoglycans

硫酸化糖胺聚糖（GAG）及其类似物，如戊聚糖多硫酸酯（PPS）、硫酸葡聚糖（DS）和肝素，可抑制朊病毒感染细胞中朊病毒蛋白（PrP^）的异常亚型<Sc>形成，并延长羊瘙痒病感染动物的潜伏期。GAG的硫酸化不完全受管制，可能的硫酸化位点随机硫酸化。这种性质阻碍了阐明参与细胞表面上的各种生物事件的GAG的基本结构。为了解决GAG在抑制PrP^形成中的结构-活性关系<Sc>，我们通过应用基于碳水化合物模块的概念的合成策略组装了一系列糖胺聚糖类似物。在由此衍生和检测的合成糖苷及其共聚物中，单体4-磺基-N-乙酰基-葡糖胺（4SGN）和两种共聚物聚-4SGN和聚-6-磺基-N-乙酰基-葡糖胺（6SGN）<Sc>以低于20 μg/ml的50%有效剂量抑制PrP β 2形成。在连续处理中，抑制作用变得更加明显。它们降低了细胞朊病毒蛋白的表达，但不影响细胞生长。结构比较表明，C-2位的N-乙酰基与C-4或C-6位的硫酸基的重合可能参与抑制PrP^的<Sc>形成。然而，无论是单体还是二聚体6SGN，但聚-6SGN显示抑制作用，表明多价构型在6SGN的作用中的重要性。这些结果表明，人工硫酸化糖苷及其聚合物不仅可用于分析GAG的结构-活性关系，而且可用于改进新的朊病毒疾病治疗化合物。

Sulfated glycosaminoglycans (GAGs) and their analogues such as pentosan polysulfate (PPS), dextran sulfate (DS), and heparin inhibit abnormal isoform of prion protein (PrP^<Sc>) formation in prion-infected cells and prolong the incubation time of scrapie-infected animals. Sulfation of GAGs is not completely regulated and possible sulfation sites are randomly sulfated. This property impedes an elucidation of fundamental structures of GAGs that are involved in diverse biological events on cell surfaces. To address the structure-activity relationship of GAGs in the inhibition of PrP^<Sc> formation, we assembled a series of glycosaminoglycans analogues by applying our synthetic strategy based on the concept of carbohydrate modules. Among the synthetic glycosides and their copolymers thus derived and examined, monomeric 4-sulfo-N-acetyl-glucosamine (4SGN), and two copolymers, poly-4SGN and poly-6-sulfo-N-acetyl-glucosamine (6SGN), inhibited PrP^<Sc> formation with 50% effective dose lower than 20 μg/ml. The inhibitory effect became more evident in consecutive treatment. They reduced the expression of cellular prion protein but did not affect cell growth. Structural comparison suggested that coincidence of N-acetyl group at C-2 with sulfate group at C-4 or C-6 might be involved in the inhibition of PrP^<Sc> formation. However, neither monomeric nor dimeric 6SGN, but poly-6SGN showed the inhibitory effect, suggesting the importance of polyvalent configuration in the effect of 6SGN. These results indicate that the artificially sulfated glycosides and their polymers are useful not only for the analysis of structure-activity relationship of GAGs but also for the improvement of new therapeutic compounds for prion diseases.