Chang Hui-Hwa, Asano Naoki, Ishii Satoshi, Ichikawa Yoshitaka, Fan Jian-Qiang
Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA.
FEBS J. 2006 Sep;273(17):4082-92. doi: 10.1111/j.1742-4658.2006.05410.x.
Gaucher disease is an autosomal recessive lysosomal storage disorder caused by the deficient activity of glucocerebrosidase. Accumulation of glucosylceramide, primarily in the lysosomes of cells of the reticuloendothelial system, leads to hepatosplenomegaly, anemia and skeletal lesions in type I disease, and neurologic manifestations in types II and III disease. We report herein the identification of hydrophilic active-site-specific chaperones that are capable of increasing glucocerebrosidase activity in the cultured fibroblasts of Gaucher patients. Screening of a variety of natural and synthetic alkaloid compounds showed isofagomine, N-dodecyl deoxynojirimycin, calystegines A3, B1, B2 and C1, and 1,5-dideoxy-1,5-iminoxylitol to be potent inhibitors of glucocerebrosidase. Among them, isofagomine was the most potent inhibitor of glucocerebrosidase in vitro, and the most effective active-site-specific chaperone capable of increasing residual glucocerebrosidase activity in fibroblasts established from Gaucher patients with the most prevalent Gaucher disease-causing mutation (N370S). Intracellular enzyme activity increased approximately two-fold after cells had been incubated with isofagomine, and the increase in glucocerebrosidase activity was both dose-dependent and time-dependent. Western blotting demonstrated that there was a substantial increase in glucocerebrosidase protein in cells after isofagomine treatment. Immunocytochemistry revealed an improvement in the glucocerebrosidase trafficking pattern, which overlaps that of lysosome-associated membrane protein 2 in Gaucher fibroblasts cultivated with isofagomine, suggesting that the transport of mutant glucocerebrosidase is at least partially improved in the presence of isofagomine. The hydrophilic active-site-specific chaperones are less toxic to cultured cells. These results indicate that these hydrophilic small molecules are suitable candidates for further drug development for the treatment of Gaucher disease.
戈谢病是一种常染色体隐性溶酶体贮积症，由葡糖脑苷脂酶活性缺乏引起。葡糖神经酰胺主要在网状内皮系统细胞的溶酶体中蓄积，导致I型疾病出现肝脾肿大、贫血和骨骼病变，以及II型和III型疾病出现神经学表现。我们在此报告了亲水性活性位点特异性伴侣分子的鉴定，这些分子能够提高戈谢病患者培养成纤维细胞中的葡糖脑苷脂酶活性。对多种天然和合成生物碱化合物的筛选显示，异法戈明、N-十二烷基脱氧野尻霉素、月光花素A3、B1、B2和C1以及1,5-二脱氧-1,5-亚氨基木糖醇是葡糖脑苷脂酶的有效抑制剂。其中，异法戈明是体外最有效的葡糖脑苷脂酶抑制剂，也是最有效的活性位点特异性伴侣分子，能够提高来自具有最常见戈谢病致病突变（N370S）的戈谢病患者的成纤维细胞中的残余葡糖脑苷脂酶活性。在用异法戈明孵育细胞后，细胞内酶活性增加了约两倍，且葡糖脑苷脂酶活性的增加呈剂量依赖性和时间依赖性。蛋白质印迹法表明，异法戈明处理后细胞中的葡糖脑苷脂酶蛋白有显著增加。免疫细胞化学显示，在用异法戈明培养的戈谢病成纤维细胞中，葡糖脑苷脂酶的运输模式有所改善，与溶酶体相关膜蛋白2的运输模式重叠，这表明在异法戈明存在的情况下，突变型葡糖脑苷脂酶的运输至少部分得到改善。亲水性活性位点特异性伴侣分子对培养细胞的毒性较小。这些结果表明，这些亲水性小分子是用于戈谢病治疗的进一步药物开发的合适候选物。
