Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening.
Optimization and validation of two miniaturized glucocerebrosidase enzyme assays for high throughput screening.
复制标题
DOI：
10.2174/138620708786734244
复制
发表时间：
2008-12
影响因子：
1.8
通讯作者：
Austin CP
中科院分区：
文献类型：
--
作者：
Urban DJ;Zheng W;Goker-Alpan O;Jadhav A;Lamarca ME;Inglese J;Sidransky E;Austin CP
葡萄糖脑苷酶(GC)催化β-葡萄糖脑苷在溶酶体中水解为葡萄糖和神经酰胺。葡萄糖脑苷酶基因(GBA)的突变导致高谢病，这是一种常染色体隐性遗传性溶酶体储存障碍。在高谢病患者中遇到的许多突变都是错义改变，可能导致这种溶酶体蛋白的错误折叠、稳定性降低和/或错误洗脱。一些GC抑制剂已被证明作为化学伴侣，稳定突变蛋白的构象，从而恢复其功能。要高通量筛选具有伴侣治疗潜力的此类化合物的小分子文库(HTS)，需要一种准确、可重复性和灵敏的分析方法。我们调整和优化了两种荧光GC酶分析方法，并将其微型化为用于HTS的1536孔板格式。两种底物4-甲基伞形花环β-D-吡喃葡萄糖苷和间苯二酚β-D-吡喃葡萄糖苷的Km值分别为768μM和33μM，发射光谱不同。两种检测方法的配对筛选有助于消除由于自身荧光或被筛选化合物的荧光猝灭而对活性的错误推断。用Lopac文库进行的测试表明，这两种方法对HTS都有很强的检测能力，并且对GC抑制剂的活性给出了类似的结果。这两种方法均可用于鉴定具有潜在治疗价值的GC激活剂和抑制剂。
Glucocerebrosidase (GC) catalyzes the hydrolysis of β-glucocerebroside to glucose and ceramide in lysosomes. Mutations in the glucocerebrosidase gene (GBA) result in Gaucher disease, an autosomal recessive lysosomal storage disorder. Many of the mutations encountered in patients with Gaucher disease are missense alterations that may cause misfolding, decreased stability and/or mistrafficking of this lysosomal protein. Some inhibitors of GC have been shown to act as chemical chaperones, stabilizing the conformation of mutant proteins and thus restoring their function. High-throughput screening (HTS) of small molecule libraries for such compounds with potential for chaperone therapy requires an accurate, reproducible and sensitive assay method. We have adapted and optimized two fluorogenic GC enzyme assays and miniaturized them into the 1536-well plate format for HTS. The two substrates, 4-methylumbelliferyl β-D-glucopyranoside and resorufin β-D-glucopyranoside, have Km values of 768 μM and 33 μM, respectively, and different emission spectra. Paired screening with the two assays helps to eliminate false inference of activity due to autofluorescence or fluorescence quenching by the screened compounds. Test screens with the LOPAC library indicated that both assays were robust for HTS, and gave comparable results for GC inhibitor activities. These two assays can be used to identify both GC activators and inhibitors with potential therapeutic value.
登录
查看更多内容
影响因子：
3.5
作者：
Ron, I;Horowitz, M
通讯作者：
Horowitz, M
影响因子：
4.8
作者：
Premkumar, L;Sawkar, AR;Sussman, JL
通讯作者：
Sussman, JL
影响因子：
14.8
作者：
Lieberman, Raquel L.;Wustman, Brandon A.;Petsko, Gregory A.
通讯作者：
Petsko, Gregory A.
影响因子：
13.5
作者：
Hays, WS;Wheeler, DE;Johnston, DE
通讯作者：
Johnston, DE
影响因子：
3.9
作者：
PETERS, SP;COYLE, P;GLEW, RH
通讯作者：
GLEW, RH