Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.
Novel patient cell-based HTS assay for identification of small molecules for a lysosomal storage disease.
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DOI：
10.1371/journal.pone.0029504
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Maegawa GH
中科院分区：
文献类型：
--
作者：
Geng H;Whiteley G;Ribbens J;Zheng W;Southall N;Hu X;Marugan JJ;Ferrer M;Maegawa GH
小分子已被确定为潜在的溶酶体储存疾病(LSD)的治疗剂，LSD是由蛋白质缺陷引起的遗传性代谢紊乱，导致溶酶体功能障碍。一些小分子有助于突变的错误折叠的溶酶体酶的折叠，否则会在内质网相关的降解中降解。最终结果是提高了缺陷酶的残留酶活性。大多数为鉴定这些分子而开发的高通量筛选(HTS)分析都是单目标生化分析。在这里，我们描述了一种基于细胞的测试，使用患者细胞系来识别在异染性脑白质营养不良(MLD)患者中发现的增强残留芳基硫酸酯酶A(ASA)活性的小分子，MLD是一种进行性神经退行性LSD。用SV40大T抗原转化原代培养的MLD患者成纤维细胞，为大规模HTS提供足够的细胞系。这些SV40转化的细胞(SV40t)显示保存了原代细胞的生化特性。用一种特殊的底物对硝基邻苯二酚硫酸盐(PNCS)，在多孔板培养的MLD患者(ASA-I179S)的原代和SV40t成纤维细胞中观察到可检测到的AsA残留活性。在高密度的1,536孔板上，使用传统的比色底物pNCS底物，建立了一种稳健的荧光ASA分析方法，其产物(PNC)在白色固体底板中起到“板荧光猝灭”的作用。当针对不同的小分子集合进行测试时，基于细胞的定量HTS检测ASA产生了强大的统计参数。这种基于细胞的检测方法可以用于其他几种LSD和遗传疾病，特别是那些依赖于传统上对检测小型化的低灵敏度底物的那些疾病。此外，这里开发的使用患者细胞的基于细胞的HTS定量分析为在疾病细胞环境中识别治疗性小分子创造了机会，在这种环境中，潜在的被破坏的通路被暴露并可用作靶点。
Small molecules have been identified as potential therapeutic agents for lysosomal storage diseases (LSDs), inherited metabolic disorders caused by defects in proteins that result in lysosome dysfunctional. Some small molecules function assisting the folding of mutant misfolded lysosomal enzymes that are otherwise degraded in ER-associated degradation. The ultimate result is the enhancement of the residual enzymatic activity of the deficient enzyme. Most of the high throughput screening (HTS) assays developed to identify these molecules are single-target biochemical assays. Here we describe a cell-based assay using patient cell lines to identify small molecules that enhance the residual arylsulfatase A (ASA) activity found in patients with metachromatic leukodystrophy (MLD), a progressive neurodegenerative LSD. In order to generate sufficient cell lines for a large scale HTS, primary cultured fibroblasts from MLD patients were transformed using SV40 large T antigen. These SV40 transformed (SV40t) cells showed to conserve biochemical characteristics of the primary cells. Using a specific colorimetric substrate para-nitrocatechol sulfate (pNCS), detectable ASA residual activity were observed in primary and SV40t fibroblasts from a MLD patient (ASA-I179S) cultured in multi-well plates. A robust fluorescence ASA assay was developed in high-density 1,536-well plates using the traditional colorimetric pNCS substrate, whose product (pNC) acts as “plate fluorescence quencher” in white solid-bottom plates. The quantitative cell-based HTS assay for ASA generated strong statistical parameters when tested against a diverse small molecule collection. This cell-based assay approach can be used for several other LSDs and genetic disorders, especially those that rely on colorimetric substrates which traditionally present low sensitivity for assay-miniaturization. In addition, the quantitative cell-based HTS assay here developed using patient cells creates an opportunity to identify therapeutic small molecules in a disease-cellular environment where potentially disrupted pathways are exposed and available as targets.
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影响因子：
2.9
作者：
Motabar O;Shi ZD;Goldin E;Liu K;Southall N;Sidransky E;Austin CP;Griffiths GL;Zheng W
通讯作者：
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DOI：
10.1016/j.ejpb.2007.01.004
发表时间：
2007-08-01
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作者：
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作者：
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