Miura Kazuki, Tsukagoshi Takumi, Hirano Takako, Nishio Toshiyuki, Hakamata Wataru
Department of Chemistry and Life Science, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-0880, Japan.
ACS Med Chem Lett. 2019 Aug 26;10(9):1309-1313. doi: 10.1021/acsmedchemlett.9b00259. eCollection 2019 Sep 12.
Inhibitors of human α-l-fucosidases, tissue α-l-fucosidase (tFuc), and plasma α-l-fucosidase reportedly play roles in multiple diseases, suggesting their therapeutic potential for gastric disease associated with and fucosidosis. Terminal fucose linkages on glycoproteins and glycolipids are a natural substrate for both enzymes; however, there are currently no fluorogenic substrates allowing their cellular evaluation. Here, we described the development of novel three-color fluorogenic substrates for lysosome-localized tFuc that exhibited excellent specificity and sensitivity in three human cell lines. Additionally, we developed a cell-based high-throughput inhibitor screening system in a 96-well format and a cell-based inhibitory activity evaluation system in a 6-well format for tFuc inhibitors using this substrate, which allowed accurate quantification of the inhibition rate. Moreover, analysis of significant changes in gene expression resulting from 30% inhibition of tFuc in HeLa cells revealed potential roles in gastric disease.
据报道，人α-L-岩藻糖苷酶、组织α-L-岩藻糖苷酶（tFuc）和血浆α-L-岩藻糖苷酶的抑制剂在多种疾病中发挥作用，表明它们对与岩藻糖苷贮积症相关的胃部疾病具有治疗潜力。糖蛋白和糖脂上的末端岩藻糖连接是这两种酶的天然底物；然而，目前尚无用于细胞评估的荧光底物。在此，我们描述了用于溶酶体定位的tFuc的新型三色荧光底物的开发，该底物在三种人类细胞系中表现出优异的特异性和灵敏度。此外，我们使用该底物开发了96孔格式的基于细胞的高通量抑制剂筛选系统和6孔格式的基于细胞的tFuc抑制剂抑制活性评估系统，能够准确量化抑制率。此外，对HeLa细胞中tFuc 30%抑制导致的基因表达显著变化的分析揭示了其在胃部疾病中的潜在作用。
